NS1-NP Diagnostics of Influenza Virus Infection

ABSTRACT

The present application describes methods for assessing influenza infection, including prognosis. An assay that determines the amount of the NS1 and NP proteins of influenza virus shows enhanced sensitivity and reliability compared to either antigen alone. Many formats employ pan-specific antibodies (i.e., that react with all or at least with multiple strains within an influenza type).

This application claims priority to U.S. Provisional App. No. 61/036,954 (filed Mar. 15, 2008), which is incorporated by reference in its entirety. In addition, International patent applications PCT/US06/26155, filed Jul. 3, 2006, PCT/US06/41748 filed Oct. 21, 2006, and PCT/US08/01123 filed on Jan. 28, 2008, as well as U.S. applications Nos. 11/698,798 filed Jan. 26, 2007; 11/481,411 filed Jul. 3, 2006, 60/792,274, filed Apr. 14, 2006, 60/765,292, filed Feb. 2, 2006, 60/726,377, filed Oct. 13, 2005; and 60/696,221, filed Jul. 1, 2005, are directed to related subject matter and each application is incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a Sequence Listing provided in ASCII text file 6610SEQLIST.txt, of size 375,278 bytes and created on Mar. 16, 2009, which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Influenza is caused by an RNA virus of the orthomyxoviridae family. There are three types of these viruses and they cause three different types of influenza: type A, B and C. Influenza virus type A viruses infect mammals (humans, pigs, ferrets, horses) and birds. This is very important to mankind, as this is the type of virus that has caused worldwide pandemics. Influenza virus type B (also known simply as influenza B) infects only humans. It occasionally causes local outbreaks of flu. Influenza type C viruses also infect only humans. They infect most people when they are young and rarely causes serious illness.

Current rapid immunodiagnostic tests for influenza antigens like the Tauns Capilia™ assay (Tauns Laboratories, Inc., Japan), “Binax NOW FluA and FluB™” (Binax, Inc., Portland, Me.), “Directigen Flu A+B™” (Becton Dickinson, Franklin Lakes, N.J.), “Flu OIA™” (Biostar Inc., Boulder, Colo.), “Quick Vue™” (Quidel, Sand Diego, Calif.), “Influ AB Quick™” (Denka Sieken Co., Ltd., Japan) and “Xpect Flu A & B” (Remel Inc., Lenexa, Kans.), can reportedly either detect influenza A or distinguish between influenza A and B. The complexity of the test formats may require special training. In addition, significant amounts of virion particles are commonly required to obtain a positive test result, limiting their use to a short window of time when virus shedding is at its highest levels. Assay sensitivity is also variable with up to 20% false negative test results in certain assays being of significant current concern (e.g., see “WHO recommendations on the use of rapid testing for influenza diagnosis,” July 2005). Reverse-transcriptase PCR-based diagnostics (RT-PCR) has resulted in advances in capabilities, but is laborious and requires highly trained personnel making on-site or field-testing difficult. Because of the relative inefficiency of the reverse transcriptase enzyme, significant amounts of virus (e.g., 10⁴ virion particles) and as many as 20 primers may be required effectively to detect viral RNA. Unfortunately, RT-PCR is not easily adapted to high throughput screening of subjects in an epidemic setting or to field uses in an agricultural or point-of-care setting.

BRIEF SUMMARY OF THE INVENTION

Among other things, the invention provides a method of determining the presence of and/or assessing an influenza virus infection, comprising: (a) determining an amount of influenza virus NS1 protein in a sample from a subject, and (b) determining an amount of influenza virus nucleoprotein (NP) in the sample, wherein a detectable amount of NS1 protein and/or NP protein indicates that the subject is infected with an influenza virus and/or a ratio of an amount of NS1 protein to an amount of NP protein indicates a prognosis of the subject.

Optionally, the method further comprises comparing the amount of NS1 with the amount of NP in the sample. Optionally, the prognosis includes stage of infection, progress of infection over time, severity of infection, outcome of infection under a treatment of interest, and/or amenability to treatment. Optionally, the method further comprises determining the stage of infection from the ratio, a relatively higher ratio indicating a relatively earlier stage of infection. Optionally, the method further comprises selecting a treatment regime from the ratio, a relatively high ratio favoring administering an anti-viral agent that inhibits viral reproduction and/or isolating the patient. Optionally, the method is performed at different times, wherein changes in the ratio with time provide an indication of the course of infection.

Optionally, the amount of NS1 and/or NP protein is determined using at least one antibody that specifically binds to NS1 or NP. Optionally, the amount of NS1 is determined using at least one NS1 antibody and the amount of NP is determined using at least one NP antibody. Optionally, determining the amount of NS1 or NP comprises: contacting the sample with at least one NS1 or NP antibody, and determining the amount of a complex of the at least antibody specifically bound to the NS1 or NP protein, wherein the amount of the complex is an indication of the amount of NS1 or NP in the sample.

Optionally, the at least one NS1 antibody or at least one NP antibody comprises an antibody that is pan-specific to influenza virus type A. Optionally, the at least one NS1 antibody competes with an antibody selected from the group consisting of F64 3H3, F68 8E6, F64 6G12, F68 10A5, F80 7E8, F80 8F6, F80 9B1, F81 1C12, F81 1F3, F81 4D5, and F64 1A10.

Optionally, determining the amount of the antigen (NS1 and/or NP) comprises contacting the sample with first and second pan specific antibodies that bind to different epitopes of the antigen; and determining the amount of a complex between the first and second antibodies and the antigen, wherein the amount of the complex is an indication of the amount of the antigen in the sample. Optionally, the method is performed using a lateral flow format.

Optionally, the pan-specific antibodies for NS1 each bind to an epitope within residues 8-21, 9-20, 29-38 or 45-49 of NS1. Optionally, the first and/or second pan-specific antibodies for NS1 comprises a mixture of antibodies. Optionally, the first and second pan-specific antibodies for NS1 each compete with one or more antibodies selected from the group consisting of F64 3H3, F68 4H9, F68 8E6, F64 6G12, F68 10A5, F80 3D5, F80 7E8, F80 8F6, F80 9B1, F81 1C12, F81 1F3, F81 4D5, F64 1A10, F89 6B5, F94 1F9 and F94 3A1 wherein the first pan-specific antibody binds to a different epitope than the second pan-specific antibody. Optionally, the first and second antibodies are pan-specific for NS1 from influenza A, wherein the first antibody competes with or is derived from the monoclonal antibody F64 3H3 and/or F68 4H9 and is optionally immobilized on a solid substrate, and the second antibody competes with or is derived from the monoclonal antibody F68 8E6 and/or F80 3D5 and is optionally gold-conjugated.

Optionally, the first and second antibodies are pan-specific for NS1 from influenza B, the first antibody competes with or is derived from the monoclonal antibody F89 6B5 and is optionally immobilized on a solid substrate, and the second antibody competes with or is derived from the monoclonal antibody F94 1F9 and/or F94 3A1 and is optionally gold-conjugated.

Optionally, the sample is a nasal or throat sample. Optionally, the subject is a human showing symptoms of influenza. Optionally, the amount of NS1 and/or NP is determined using at least one PDZ polypeptide that binds specifically to NS1 or NP. Optionally, the PDZ polypeptide is immobilized to a solid phase and the NS1 and/or NP antibody is a detection antibody. Optionally, the at least one PDZ polypeptide that binds specifically to NS1 is selected from the group consisting of PSD95 domain 2, INADL domain 8, or combinations thereof.

Optionally, the method is performed on samples from a test subject and a control subject, the test subject being treated with a test agent, and the control subject being untreated with the test agent, and the method further comprises comparing the change in the NS1:NP ratio in the two subjects over time. A quicker decrease in NS1:NP ratio over time in one or more test subjects kept in the presence of the test agent can indicate that the test agent is effective in treating influenza during the early stages of infection.

Optionally, the amount of NS1 and/or NP and/or the NS1:NP ratio in the sample(s) (“test sample(s)”) is compared to that measured in a control sample. The control sample can be taken from a control subject believed to be infected with influenza virus. The control subject for example at a known (or believed) stage of infection. Examples of subjects at an early stage of infection include subjects infected within the last 72 hours, e.g., within the last 48 or 24 hours. Examples of subjects at a late stage of infection include those infected at least 5 days ago, e.g., to have been infected at least 1 week ago.

Optionally, the amount of NS1 and/or NP and/or the NS1:NP ratio in the sample(s) is compared to that measured in multiple control samples taken at different timepoints of an influenza virus infection in a control subject. For example, the amount of NS1 and/or NP in the test sample(s) and control sample(s) is measured by contacting the test sample(s) and control sample(s) with the same solution of an NS1-binding agent and/or an NP-binding agent. The amount of NS1 and/or NP and/or the NS1:NP ratio in the sample(s) can be measured using a lateral flow assay in which an NS1-capture agent and an NP-capture agent are both immobilized within the same area of a solid support.

Optionally, the method further comprises determining an amount of an NA, and/or an HA protein and/or an M1 protein the sample, wherein a detectable amount of NA protein and/or HA protein and/or M1 protein indicates the subject is infected with influenza virus. Optionally, the method further comprises determining a subtype of and NA protein and/or an HA protein and/or type of M1 protein or an NS1 protein or and M1 protein in the sample, wherein the subtype of HA or NA protein indicates the strain of the influenza virus, and the type of the NS1 protein and/or NP protein and/or M1 protein indicates the type of influenza virus the subject is infected with.

Optionally, the method further comprises contacting the sample with first and second PDZ domains, and the method further comprises determining relative binding of the first and second PDZ domains to NS1 protein the sample and typing the influenza virus infection as pathogenic or nonpathogenic from the relative binding. Optionally, the first and second PDZ domains are PSD95 and INADL.

Optionally, determining an amount of influenza virus antigen (e.g., NS1 and/or NP) in a sample comprises contacting the sample with first and second binding agents specific for the influenza A and influenza B type of that antigen, and the method further comprises determining relative binding to the first and second binding agents, and characterizing the influenza virus infection as A or B based on the relative binding.

The invention further provides an array comprising at least one first agent that binds to influenza virus NS1 and at least one agent that binds to NP. Optionally, at least one NS1-binding agent and/or at least one NP-binding agent is an antibody or a PDZ polypeptide. Optionally, the array comprises at least two pan-specific NS1 antibodies and/or at least two pan-specific NP antibodies. Optionally, the array comprises at least two subtype-specific NS1 antibodies and/or at least two subtype-specific NP antibodies. Optionally, the array comprises at least one antibody specific for influenza A NS1 and at least one antibody specific for influenza B NS1. Optionally, the array comprises at least one antibody specific for influenza A NP and at least one antibody specific for influenza B NP. Optionally, the array further comprises at least one agent that binds to an M1 protein and at least one agent that binds to an HA protein.

The invention also provides a kit for the assessment of an influenza virus infection in a subject, comprising: (a) a first agent for determining the amount of influenza virus NS1 protein, and (b) a second agent for determining the amount of influenza virus NP protein. Optionally the first agent or the second agent comprises at least one antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (SEQ ID NO:998) shows the invariant amino acid residues between NS1 proteins from three subtypes of influenza A: H1N1, H3N2 and H5N1. As described below, segments of NS1 protein including clusters of invariant amino acid residues are useful for inducing pan-specific antibodies.

FIG. 1B (SEQ ID NO:999) shows amino acid residues found in the NS1 protein of H5N1 but not found in H3N2 or H1N1. Clusters of these residues, particularly the clusters at positions 21-28 and at the C-terminus, are useful for preparing an antibody that binds to H5N1 without binding to the other two subtypes.

FIG. 2 (SEQ ID NO:1000) shows a consensus sequence of residues of the NS1 protein from different strains of influenza A.

FIG. 3 (SEQ ID NO:1001) shows a consensus sequence of residues of the NS1 protein form different strains of influenza B. Underlined residues are invariable between different strains.

FIG. 4 shows the results of testing nasal secretions from six human Flu A positive samples.

FIG. 5 shows NS1 expression in MDCK cells infected with A/PR/8/34.

FIG. 6 shows that PDZ interacts with NS1 in cells.

FIG. 7 shows that INADL d8 interacts with H3N2 NS1 in cells.

FIG. 8 shows a lateral flow format for an NS1 diagnostic using a PDZ capture agent and monoclonal antibody detect agent AU-4B2.

FIG. 9 shows a lateral flow format using a monoclonal antibody capture agent and a monoclonal antibody detect agent AU-4B2.

FIG. 10A-F exemplary lateral flow Influenza test formats.

FIG. 11: Detection of recombinant NS1 from two strains of influenza B in a lateral flow assay using various combinations of capture and detection antibody.

FIG. 12: Detection of NS1 from influenza B in clinical samples.

FIG. 13: Chart showing suitable combinations of capture and detection antibody for detection of NS1 from influenza B.

FIG. 14: Sequence of a GST fusion peptide comprising 3 copies of PSD95 domain 2 (SEQ ID NO: 1002). GST-derived sequence, including GST peptide sequence and cloning linker sequence, is italicized (amino acids 1-242 and 243-244 respectively). Native PSD95 domain 2 sequence is in bold (corresponding to amino acids 197 to 288 of NCBI Acc. No. AAC52113); native PSD95 sequence other than domain 2 is shown in normal (i.e., non-bold, non-italicized) font (any such sequence that is repeated/relocated is also underlined).

FIG. 15: Diagnostic testing of three diluted clinical samples for NS1 and NP antigens: results after 6, 15 and 30 minutes.

FIG. 16: Diagnostic testing of eight diluted clinical samples for NS1 and NP antigens: results after 15 minutes.

FIG. 17: Diagnostic testing of three diluted clinical samples for NS1 and NP antigens: results after 6, 15 and 30 minutes.

FIG. 18: Schematic of a NS1/NP lateral flow test format. The left and middle panels depict a format in which the NS1-capture agent and the NP-capture agent are deposited as separate lines, with the NS1 capture line situated between the NP capture line and the control line (left panel) or vice versa (middle panel). The right panel depicts a format in which the NS1-capture agent and the NP-capture agent are deposited together as a single line.

FIG. 19: Log ratio of NS1:NP over course of infection using throat samples from three patients.

FIG. 20: Log ratio of NS1:NP over course of infection using nasal samples from three patients.

FIG. 21: CAMAG absorbance unit values for AVC Flu A/B Tests (NS1) and AVC Flu A NP Tests of patients as a function of time.

FIG. 22: Summary of influenza B virus detection in human clinical nasal samples in a “double-line” lateral flow format.

DEFINITIONS

“Avian influenza A” means an influenza A subtype that infects an avian subject and is transmissible between avian subjects. Representative examples of avian influenza hemmagglutinin subtypes include H5, H6, H7, H9 and H10 and representative strains include H5N1, H6N2, H7N3, H7N7, H9N2, H10N4 and H10N5. Some strains of Avian influenza can also infect humans.

“Avian subject” means a subject suitable for testing or treatment including all species of birds, including both wild birds (such as wildfowl) and domesticated species (such as poultry). Preferably, the avian subject to be tested or treated is selected from the group consisting of chickens, turkeys, ducks, geese, quail, ostrich, emus and exotic birds such as parrots, cockatoos and cockatiels. More preferably, the avian subject to be tested is a chicken, turkey, goose or quail.

“Pathogenic strain of influenza A” when used in the context of distinguishing between different strains of influenza virus means a “notifiable avian influenza” (NAI) virus according to the guidelines set forth by the OIE World Organization for Animal Health, World Health Organization or their designated representatives e.g., as set forth in the OIE “Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edition, 2004 (www.oie.int). Further, the subject pathogenic strain has “high pathogenicity” in a representative test for virulence or an H5 or H7 virus with an influenza A hemmagglutinin (HA) precursor protein HA0 cleavage site amino acid sequence that is similar to any of those that have been observed in virulent viruses, i.e., as defined by the OIE or a representative similar national or international organization or trade association. Representative examples of HA0 cleavage site amino acid sequences in virulent H5 and H7 strains of influenza A comprise multiple basic amino acids (arginine or lysine) at the cleavage site of the viral precursor hemagglutinin protein, e.g., where low virulence strains of H7 viruses have PEIPKGR*GLF (SEQ ID NO:20) or PENPKGR*GLF (SEQ ID NO:21) highly pathogenic strains have -PEIPKKKKR*GLF (SEQ ID NO:22), PETPKRKRKR*GLSF (SEQ ID NO:23), PEIPKKREKR*GLF (SEQ ID NO:24) or PETPKRRRR*GLF (SEQ ID NO:25). Current representative tests for virulence include inoculation of 4-8 week old chickens with infectious virus wherein strains are considered to be highly pathogenic if they cause more than 75% mortality within 10 days; and/or, any virus that has an intravenous pathogenicity index (IVPI) greater than 1.2, wherein intravenously inoculated birds are examined at 24-hour intervals over a 10-day period; scored for “0”, normal; “1” sick; “2” severely sick”; “3” dead; and, the mean score calculated as the IVPI. The latter highly pathogenic strains are referred to by the OIE as a “highly pathogenic NAI virus” (HPNIA). Current representative examples of NAI include the H5 and H7 strains of influenza A. Current representative examples of HPNIA include H5N1.

“Less Pathogenic strain of influenza A” means an avian influenza A that is notifiable, i.e., an NAI isolate (supra), but which is not pathogenic for chickens and does not have an HA0 cleavage site amino acid sequence similar to any of those that have been observed in virulent viruses, i.e., a strain referred to by the OIE as a “low pathogenicity avian influenza (LPAI).

Strains of influenza A that are not classified as highly pathogenic or less pathogenic are referred to as seasonal flu. Most strains of influenza A H1N1 are seasonal flu. However, one strain responsible for the 1918 Spanish flu is highly pathogenic.

“PDZ domain” means an amino acid sequence homologous over about 90 contiguous amino acids; preferably about 80-90; more preferably, about 70-80, more preferably about 50-70 amino acids with the brain synaptic protein PSD95, the Drosophila septate junction protein Discs-Large (DLG) and/or the epithelial tight junction protein ZO1 (ZO1). Representative examples of PDZ domains are also known in the art as Discs-Large homology repeats (“DHRs”) and “GLGF” repeats (SEQ ID NO:26). Examples of PDZ domains are found in diverse membrane-associated proteins including members of the MAGUK family of guanylate kinase homologs, several protein phosphatases and kinases, neuronal nitric oxide synthase, tumor suppressor proteins, and several dystrophin-associated proteins, collectively known as syntrophins. The instant PDZ domains encompass both natural and non-natural amino acid sequences. Representative examples of PDZ domains include: (a) polymorphic variants of PDZ proteins, (b) chimeric PDZ domains containing portions of two different PDZ proteins or a PDZ-derived portion and a non-PDZ portion, (c) fragments of a PDZ domain that are capable of binding specifically to a cognate PL, and the like. A “PDZ polypeptide” is any peptide derived from a PDZ domain, including sequence variants, chimeric polypeptides containing portions of two different PDZ proteins or a PDZ-derived portion and a non-PDZ portion, fragments of a PDZ domain that are capable of binding specifically to a cognate PL, and the like. Preferably, the instant PDZ domains contain amino acid sequences which are substantially identical to those disclosed in U.S. patent application Ser. No. 10/485,788 (filed Feb. 3, 2004), International patent application PCT/US03/285/28508 (filed Sep. 9, 2003), International patent application PCT/US01/44138 (filed Nov. 9, 2001), incorporated herein by reference in their entirety. Representative non-natural PDZ domains include those in which the corresponding genetic code for the amino acid sequence has been mutated, e.g., to produce amino acid changes that alter (strengthen or weaken) either binding or specificity of binding to PL. Optionally a PDZ domain or a variant thereof has at least 50, 60, 70, 80 or 90% sequence identity with a PDZ domain from at least one of brain synaptic protein PSD95, the Drosophila septate junction protein Discs-Large (DLG) and/or the epithelial tight junction protein ZO1 (ZO1), and animal homologs. Optionally a variant of a natural PDZ domain has at least 90% sequence identity with the natural PDZ domain. Sequence identities of PDZ domains are determined over at least 70 amino acids within the PDZ domain, preferably 80 amino acids, and more preferably 80-90 or 80-100 amino acids. Amino acids of analogs are assigned the same numbers as corresponding amino acids in the natural human sequence when the analog and human sequence are maximally aligned. Analogs typically differ from naturally occurring peptides at one, two or a few positions, often by virtue of conservative substitutions. The term “allelic variant” is used to refer to variations between genes of different individuals in the same species and corresponding variations in proteins encoded by the genes. An exemplary PDZ domain for PSD95 d2 is provided as SEQ ID NO:1.

“PDZ protein”, used interchangeably with “PDZ-domain containing polypeptides” and “PDZ polypeptides”, means a naturally occurring or non-naturally occurring protein having a PDZ domain (supra). Representative examples of PDZ proteins have been disclosed previously (supra) and include CASK, MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, TIP-43, LDP, LIM, LIMK1, LIMK2, MPP2, AF6, GORASP1, INADL, KIAA0316, KIAA1284, MAGI1, MAST2, MINT1, NSP, NOS1, PAR3, PAR3L, PAR6 beta, PICK1, Shank 1, Shank 2, Shank 3, SITAC-18, TIP1, and ZO-1. The instant non-natural PDZ domain polypeptides useful in screening assays may contain e.g. a PDZ domain that is smaller than a natural PDZ domain. For example a non-natural PDZ domain may optionally contain a “GLGF” motif, i.e., a motif having the GLGF amino acid sequence (SEQ ID NO:26), which typically resides proximal, e.g. usually within about 10-20 amino acids N-terminal, to an PDZ domain. The latter GLGF motif (SEQ ID NO:26), and the 3 amino acids immediately N-terminal to the GLGF motif (SEQ ID NO:26) are often required for PDZ binding activity. Similarly, non-natural PDZ domains may be constructed that lack the n-sheet at the C-terminus of a PDZ domain, i.e., this region may often be deleted from the natural PDZ domain without affecting the binding of a PL. Some exemplary PDZ proteins are provided and the GI or accession numbers are provided in parenthesis: PSMD9 (9184389), af6 (430993), AIPC (12751451), ALP (2773059), APXL-1 (13651263), MAGI2 (2947231), CARDI1 (1282772), CARDI4 (13129123), CASK (3087815), CNK1 (3930780), CBP (3192908), Densin 180 (16755892), DLG1 (475816), DLG2 (12736552), DLG5 (3650451), DLG6 splice var 1 (14647140), DLG6 splice var 2 (AB053303), DVL1 (2291005), DVL2 (2291007), DVL3 (6806886), ELFIN 1 (2957144), ENIGMA (561636), ERBIN (8923908), EZRIN binding protein 50 (3220018), FLJ00011 (10440342), FLJ11215 (11436365), FLJ12428 (BC012040), FLJ12615 (10434209), FLJ20075 Semcap2 (7019938), FLJ21687 (10437836), FLJ31349 (AK055911), FLJ32798 (AK057360), GoRASP1 (NM031899), GoRASP2 (13994253), GRIP1 (4539083), GTPase Activating Enzyme (2389008), Guanine Exchange Factor (6650765), HEMBA 1000505 (10436367), HEMBA 1003117 (7022001), HSPC227 (7106843), HTRA3 (AY040094), HTRA4 (AL576444), INADL (2370148), KIAA0147 Vartul (1469875), KIAA0303 MAST4 (2224546), KIAA0313 (7657260), KIAA0316 (6683123), KIAA0340 (2224620), KIAA0380 (2224700), KIAA0382 (7662087), KIAA0440 (2662160), KIAA0545 (14762850), KIAA0559 (3043641), KIAA0561 MAST3 (3043645), KIAA0613 (3327039), KIAA0751 RIM2 (12734165), KIAA0807 MAST2 (3882334), KIAA0858 (4240204), KIAA0902 (4240292), KIAA0967 (4589577), KIAA0973 SEMCAP3 (5889526), KIAA1202 (6330421), KIAA1222 (6330610), KIAA1284 (6331369), KIAA1389 (7243158), KIAA1415 (7243210), KIAA1526 (5817166), KIAA1620 (10047316), KIAA1634 MAGI3 (10047344), KIAA1719 (1267982), LIM Mystique (12734250), LIM (3108092), LIMK1 (4587498), LIMK2 (1805593), LIM-RIL (1085021), LU-1 (U52111), MAGI1 (3370997), MGC5395 (BC012477), MINT1 (2625024), MINT3 (3169808) MPP1 (189785), MPP2 (939884), MPP3 (1022812), MUPP1 (2104784), NeDLG (10853920), Neurabin II (AJ401189), NOS1 (642525), novel PDZ gene (7228177), Novel Serine Protease (1621243), Numb Binding Protein (AK056823), Outer Membrane Protein (7023825), p55T (12733367), PAR3 (8037914), PAR3-like (AF428250), PARE (2613011), PAR6BETA (13537116), PAR6GAMMA (13537118), PDZ-73 (5031978), PDZK1 (2944188), PICK1 (4678411), PIST (98394330), prIL16 (1478492), PSAP (6409315), PSD95 (3318652), PTN-3 (179912), PTN-4 (190747), PTPL1 (515030), RGS12 (3290015), RGS3 (18644735), Rho-GAP10 (NM020824), Rhophilin-like (14279408), Serine Protease (2738914), Shank 2 (6049185), Shank 3 (AC000036), Shroom (18652858), Similar to GRASP65 (14286261), Similar to Ligand of Numb px2 (BC036755), Similar to PTP Homolog (21595065), SIP1 (2047327), SITAC-18 (8886071), SNPCIIA (20809633), Shank 1 (7025450), Syntenin (2795862), Syntrophin 1 alpha (1145727), Syntrophin beta 2 (476700), Syntrophin gamma 1 (9507162), Syntrophin gamma 2 (9507164), TAX2-like protein (3253116), TIAM 1 (4507500), TIAM 2 (6912703), TIP 1 (2613001), TIP2 (2613003), TIP33 (2613007), TIP43(2613011), X-11 beta (3005559), ZO-1 (292937), ZO-2 (12734763), ZO-3 (10092690).

“PDZ ligand”, abbreviated “PL”, means a naturally occurring protein that has an amino acid sequence which binds to and forms a molecular interaction complex with a PDZ-domain. Representative examples of PL have been provided previously in prior US and International patent applications (supra).

“Specific binding” between a binding agent, e.g., an antibody or a PDZ domain, and an NS1 protein refers to the ability of a capture- or detection-agent to preferentially bind to a particular viral analyte that is present in a mixture of different viral analytes The particular analyte is for example an influenzaviral analyte in a mixture of other influenzaviral and/or non-influenzaviral analytes. Analgous considerations apply to other analytes. A binding agent preferentially binds to a particular analyte in a mixture of analytes, some of which may be present in excess compared to the particular analyte. Optionally, the agent binds specifically to the particular analyte at least 2×, 5×, 10×, 30×, 100, 300× or 1000× higher affinity than unrelated control proteins. Specific binding is often the result of interaction between specific surface structure of the binding agent and/or ligand (e.g., hydrogen bonds), whereas non-specific binding is relatively independent of specific surface structures (e.g., van der Waals forces). Specific binding to a particular analyte over unrelated proteins may or may not mean that that a binding agent preferentially binds to the analyte over closely related analytes. Some binding agents are type- or subtype-specific, e.g., the agent binds preferentially to a protein from influenzavirus of a given type or subtype over the same protein from influenzavirus of a different type or subtype. Other binding agents bind multiple types or subtypes of analytes. For example, some antibodies described in the application specifically bind to NS1 from influenza B without specifically binding to NS1 from influenza A, and vice versa. Specific binding also means a dissociation constant (KD) that is less than about 10⁻⁶M; preferably, less than about 10⁻⁷M; and, most preferably, less than about 10⁻⁸ M, In some methods, specific binding interaction is capable of discriminating between proteins having or lacking a PL with a discriminatory capacity greater than about 10- to about 100-fold; and, preferably greater than about 1000- to about 10.000-fold. Specific binding can readily be distinguished from nonspecific binding, by for example, by subtracting from a test signal a background signal from a control sample known to lack analyte and/or by generating a signal from binding of two binding agents to different epitopes of the same analyte as in a sandwich assay.

“Capture agent/analyte complex” is a complex that results from the specific binding of a capture agent, with an analyte, e.g. an influenza viral NS1 protein. A capture agent and an analyte specifically bind, i.e., the one to the other, under conditions suitable for specific binding, wherein such physicochemical conditions are conveniently expressed e.g. in terms of salt concentration, pH, detergent concentration, protein concentration, temperature and time. The subject conditions are suitable to allow binding to occur e.g. in a solution; or alternatively, where one of the binding members is immobilized on a solid phase. Representative conditions so-suitable are described in e.g., Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989). Suitable conditions preferably result in binding interactions having dissociation constants (KD) that are less than about 10⁻⁶M; preferably, less than about 10⁻⁷M; and, most preferably less than about 10⁻⁸M.

“Solid phase” means a surface to which one or more reactants may be attached electrostatically, hydrophobically, or covalently. Representative solid phases include e.g.: nylon 6; nylon 66; polystyrene; latex beads; magnetic beads; glass beads; polyethylene; polypropylene; polybutylene; butadiene-styrene copolymers; silastic rubber; polyesters; polyamides; cellulose and derivatives; acrylates; methacrylates; polyvinyl; vinyl chloride; polyvinyl chloride; polyvinyl fluoride; copolymers of polystyrene; silica gel; silica wafers glass; agarose; dextrans; liposomes; insoluble protein metals; and, nitrocellulose. Representative solid phases include those formed as beads, tubes, strips, disks, filter papers, plates and the like. Filters may serve to capture analyte e.g. as a filtrate, or act by entrapment, or act by covalently binding. A solid phase capture reagent for distribution to a user may consist of a solid phase coated with a “capture reagent”, and packaged (e.g., under a nitrogen atmosphere) to preserve and/or maximize binding of the capture reagent to an influenza NS1 analyte in a biological sample.

Samples include tissue fluids, tissue sections, biological materials carried in the air or in water and/or collected there from e.g. by filtration, centrifugation and the like, e.g., for assessing bioterror threats and the like. Alternative biological samples can be taken from fetus or egg, egg yolk, and amniotic fluids. Representative biological fluids include urine, blood, plasma, serum, cerebrospinal fluid, semen, lung lavage fluid, feces, sputum, mucus, water carrying biological materials and the like, as well as material taken or derived from these biological fluids. Alternatively, biological samples include nasopharyngeal or oropharyngeal swabs, nasal lavage fluid, tissue from trachea, lungs, air sacs, intestine, spleen, kidney, brain, liver and heart, sputum, mucus, water carrying biological materials, cloacal swabs, sputum, nasal and oral mucus, and the like. Samples can be analyzed with or without further processing. Further processing can add or remove materials present in a sample as originally removed from a subject. Representative biological samples also include foodstuffs, e.g., samples of meats, processed foods, poultry, swine and the like. Biological samples also include contaminated solutions (e.g., food processing solutions and the like), swab samples from out-patient sites, hospitals, clinics, food preparation facilities (e.g., restaurants, slaughter houses, cold storage facilities, supermarket packaging and the like). Biological samples may also include in situ tissues and bodily fluids (i.e., samples not collected for testing), e.g., the instant methods may be useful in detecting the presence or severity or viral infection in the eye e.g., using eye drops, test strips applied directly to the conjunctiva; or, the presence or extent of lung infection by e.g. placing an indicator capsule in the mouth or nasopharynx of the test subject. Alternatively, a swab or test strip can be placed in the mouth. The biological sample may be derived from any tissue, organ or group of cells of the subject. In some embodiments a scrape, biopsy, or lavage is obtained from a subject. Biological samples may include bodily fluids such as blood, urine, sputum, and oral fluid; and samples such as nasal washes, swabs or aspirates, tracheal aspirates, chancre swabs, and stool samples, or materials derived from such samples. Methods are known to those of skill in the art for the collection of biological specimens suitable for the detection of individual pathogens of interest, for example, nasopharyngeal specimens such as nasal swabs, washes or aspirates, or tracheal aspirates in the case of high risk influenza A viruses involved in respiratory disease, oral swabs and the like. Optionally, the biological sample may be suspended in an isotonic solution containing antibiotics such as penicillin, streptomycin, gentamycin, and mycostatin.

“Isolated” or “purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises a significant percent (e.g., greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%-100%) of the sample in which it resides. In certain embodiments, a substantially purified component comprises at least 50%, 80%-85%, or 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density. Generally, a substance is purified when it exists in a sample in an amount, relative to other components of the sample that is not found naturally.

“Subject”, is used herein to refer to an organism such as a human or to an animal such as a domesticated animal, e.g. mammals, fishes, birds, reptiles, amphibians and the like. If so desired, the subject can be cells maintained in vitro or ex vivo (for example cultured or isolated or recombinant cells infected with influenza virus). Some subjects have influenza or at least one symptom thereof. Some subjects are at increased risk of influenza, e.g., previously exposed to influenza virus or to another organism suspected to have influenza. Some subjects not exhibit any symptom of influenza, or not suspected of having influenza, or is not at increased risk for influenza.

“Signal generating compound”, abbreviated “SGC”, means a molecule that can be linked to an antibody or a PL or a PDZ (e.g. using a chemical linking method as disclosed further below and is capable of reacting to form a chemical or physical entity (i.e., a reaction product) detectable in an assay according to the instant disclosure. Representative examples of reaction products include precipitates, fluorescent signals, compounds having a color, and the like. Representative SGC include e.g., bioluminescent compounds (e.g., luciferase), fluorophores (e.g., below), bioluminescent and chemiluminescent compounds, radioisotopes (e.g., ¹³¹I, ¹²⁵I, ¹⁴C, ³H, ³⁵S, ³²P and the like), enzymes (e.g., below), binding proteins (e.g., biotin, avidin, streptavidin and the like), magnetic particles, chemically reactive compounds (e.g., colored stains), labeled oligonucleotides; molecular probes (e.g., CY3, Research Organics, Inc.), and the like. Representative fluorophores include fluorescein isothiocyanate, succinyl fluorescein, rhodamine B, lissamine, 9,10-diphenlyanthracene, perylene, rubrene, pyrene and fluorescent derivatives thereof such as isocyanate, isothiocyanate, acid chloride or sulfonyl chloride, umbelliferone, rare earth chelates of lanthanides such as Europium (Eu) and the like. Representative SGC's useful in a signal generating conjugate include the enzymes in: IUB Class 1, especially 1.1.1 and 1.6 (e.g., alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and the like); IUB Class 1.11.1 (e.g., catalase, peroxidase, amino acid oxidase, galactose oxidase, glucose oxidase, ascorbate oxidase, diaphorase, urease and the like); IUB Class 2, especially 2.7 and 2.7.1 (e.g., hexokinase and the like); IUB Class 3, especially 3.2.1 and 3.1.3 (e.g., alpha amylase, cellulase, β-galacturonidase, amyloglucosidase, β-glucuronidase, alkaline phosphatase, acid phosphatase and the like); IUB Class 4 (e.g., lyases); IUB Class 5 especially 5.3 and 5.4 (e.g., phosphoglucose isomerase, trios phosphatase isomerase, phosphoglucose mutase and the like.) Signal generating compounds also include SGC whose products are detectable by fluorescent and chemiluminescent wavelengths, e.g., luciferase, fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series; compounds such as luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds such as luciferin; fluorescent proteins; and the like. Fluorescent proteins include, but are not limited to the following: namely, (i) green fluorescent protein (GFP), i.e., including, but not limited to, a “humanized” versions of GFP wherein codons of the naturally-occurring nucleotide sequence are exchanged to more closely match human codon bias; (ii) GFP derived from Aequoria victoria and derivatives thereof, e.g., a “humanized” derivative such as Enhanced GFP, which are available commercially, e.g., from Clontech, Inc.; (iii) GFP from other species such as Renilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle et al. (2001) J. Protein Chem. 20:507-519; (iv) “humanized” recombinant GFP (hrGFP) (Stratagene); and, (v) other fluorescent and colored proteins from Anthozoan species, such as those described in Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like. The subject signal generating compounds can be coupled to a PL or PDZ domain polypeptide. SGCs can also be attached to any agent of interest, e.g., antibodies. Attaching certain SGC to agents can be accomplished through metal chelating groups such as EDTA. The subject SGC share the common property of allowing detection and/or quantification of an influenza PL analyte in a test sample. The subject SGC are detectable using a visual method; preferably, with a method amenable to automation such as a spectrophotometric method, a fluorescence method, a chemiluminescent method, a electrical nanometric method involving e.g., a change in conductance, impedance, resistance and the like and a magnetic field method. Some SGC's are detectable with the naked eye. Some SGC's are detectable with a signal detection apparatus, such as those described herein. Some SGCs are not themselves detectable but become detectable when subject to further treatment with e.g.,.

The SGC can be attached in any manner (e.g., through covalent or non-covalent bonds) to a binding agent of interest (e.g., an antibody or a PDZ polypeptide). SGCs suitable for attachment to agents such as antibodies include colloidal gold, fluorescent antibodies, Europium, latex particles, and enzymes. The agents that bind to NS1 and NP can each comprise distinct SGCs. For example, red latex particles can be conjugated to anti-NS1 antibodies and blue latex particles can be conjugated to anti-NP antibodies. Other detectable SGCs suitable for use in a lateral flow format include any moiety that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical, or other means. For example, suitable SGCs include biotin for staining with labeled streptavidin conjugate, fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric SGCss such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex beads). Patents that described the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. See also Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.). Radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic SGCs are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

Determining the presence and/or amount of an analyte such as influenza NS1 or NP can be achieved by determining the presence and/or amount of specific binding between a binding agent and the analyte. The binding agent for example comprises an antibody or a PDZ domain that can bind specifically to a site on the analyte (e.g., influenza NS1 or NP). Suitable binding sites on the analyte include an epitope or a C-terminal PL site. Detection can be achieved in many ways. The presence and/or amount of NS1 or NP in a sample can be determined with or without immuno-PCR amplification. Immuno-PCR amplification is a technique in which a protein immunogen is detected using a binding agent labeled with a nucleic acid. PCR amplification of the nucleic acid with labeled primers which are not attached to the binding agent generates a detectable amount labeled PCR amplification product. The amount of labeled amplification product is amplified through multiple rounds of amplification in exponential fashion, where the rate of amplification of the is influenced by the amount of PCR product present, which itself increases during the course of each round of amplification. Although immuno-PCR provides a sensitive means to detect NS1 or NP, either antigen can be present at sufficient amounts to be detected by less sensitive but more simple-to-perform methods.

Optionally, detection is achieved through the use of a SGC that itself emits or can generate a detectable signal. Optionally, the SGC is attached to (or attachable to) an agent that can bind specifically to an analyte of interest. The agent is for example an antibody or a PDZ polypeptide that can bind specifically to influenza NS1 or NP. The SGC can be directly attached (or attachable) to the agent. The SGC can also be attached or attachable to a second agent that can attach to the first agent (e.g., an antibody to an anti-NS1 antibody). Optionally, the second agent specifically binds to the first agent. In an example, the first agent is first allowed to bind specifically to the analyte of interest, and a second agent is then allowed to bind specifically to the agent, where the second agent optionally comprised the SGC, or is attachable to the SGC. In other examples, the SGC is not attached or attachable to the agent.

Optionally, the SGC itself can emit or can generate a detectable signal. In an example, the SGC intrinsically emits a detectable signal, so that the presence and/or amount of the SGC can be directly determined (i.e., the presence and/or amount of the SGC need not be not inferred by determining the presence and/or amount of another detectable molecule). Optionally, the SGC can conditionally emit a detectable signal under certain conditions (e.g., fluorescent light). Optionally, the SGC does not emit a detectable signal but can be modified to emit a detectable signal. For example, the SGC does not emit a detectable signal when originally attached to the agent but can be modified later to emit a detectable signal. Optionally, the SGC converts or allows the conversion of another substance (substrate) to a product that emits a detectable signal that is not emitted by the substrate itself Optionally, the SGC can undergo a detectable change or enable a detectable change in another molecule. For example, the SGC can catalyze or co-catalyze a detectable reaction, or act as an activator or inhibitor of a detectable reaction. An SGC can for example comprise an enzyme, a coenzyme, a cofactor, a peptide, or protein or an inorganic molecule. Optionally, the SGC does not comprise a nucleic acid. Optionally, the presence of the SGC is not detected using PCR amplification if the SGC comprises a nucleic acid.

The term “label” is synonymous with SGC. A label (or SGC) optionally emits a detectable signal by itself, e.g., under proper conditions. A labeled agent includes an agent which is attached to a label so that the presence and/or amount of the agent can be determined by detecting or measuring the detectable signal emitted by the SGC (without having to determine the presence and/or amount of another detectable molecule).

Optionally, the presence and/or amount of a detectable entity can be amplified for greater sensitivity. Optionally, the signal amplification is increased in non-exponential fashion, e.g., the rate of signal amplification does not change depending upon the current amount of the signal present. Optionally, the signal amplification is achieved by more than one round of amplification using the same amplification method. Optionally, the amplification does not involve PCR amplification if the detectable entity comprises a nucleic acid.

The binding site on an analyte includes the region of the analyte to which a binding agent specifically binds and/or at least partially prevents specific binding between the analyte and other binding agents. Binding sites include epitopes of a monoclonal antibody (mAb) and PL sites recognized by a PDZ polypeptide. For example, two antibodies bind to the same or overlapping epitope if one competitively inhibits (blocks) binding of a prototypical antibody defining the competition group to the antigen (an NS1 protein of influenza A or influenza B, in the assays below). That is, a 3-fold or 5-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay compared to a control lacking the competing antibody (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990, which is incorporated herein by reference). Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Conversely, two binding agents have different binding sites on an analyte if the specific binding of one agent to the analyte has no inhibitory effect on the specific binding of the other agent to the analyte.

DETAILED DESCRIPTION OF THE INVENTION I. General Introduction

The present application in part provides methods for assessing an influenza virus infection, including infection with influenza A and/or influenza B. Existing assays are generally based upon the detection of a single influenza antigen within a sample of interest. The present inventors have found that the amounts of influenza virus antigens such as non-structural protein 1 (“NS1”) and nucleoprotein (NP), as described below, can vary widely in samples from infected subjects. For example, the amounts of NS1 and/or NP can vary between different infected subjects and can also vary within the same infected subject over time. As an example, a sample taken from an infected subject can have higher amounts of NS1 than NP, while another sample taken from the same subject (e.g., at a different timepoint) or taken from a different infected subject can by contrast have higher amounts of NP than NS1. Accordingly, the invention provides a method of assessment of an influenza virus infection in a subject, by determining the amounts of both NS1 and NP in a sample. The amount of NS1 and/or NP can be measured by any method, including those discussed herein.

The “amount” of an analyte such as NS1 or NP need not be a precise, actual or absolute measure of the total amount of analyte present. For example, a relative or comparative estimate of the level or concentration of an analyte can be sufficient. The relative amount can optionally be determined by comparison to the same analyte in a different sample, or by comparison to a different analyte in the same sample, or by comparison to a different analyte in a different sample, or by comparison to a control containing a known amount of the analyte. An amount can be a quantitative or qualitative measurement. A quantitative measurement can be relative or absolute. A qualitative measurement indicates whether or not an analyte is present at a detectable level. An analyte is present at a detectable level if a higher signal (with appropriate allowance for experimental error) is generated in a sample relative to a control in which the analyte is absent. Optionally, the signal from a positive sample is at least about 1.5× higher than background, e.g., about 2×, 5×, 10×, 30×, 100×, 300× or 1000× higher, The observed presence of either NS1 or NP or both indicates that the subject is infected with influenza virus.

The amounts of NS1 and NP can be measured by contacting the sample with a combination of a NS1-binding agent and an NP-binding agent, and measuring the specific binding between the sample and the NS1-binding agent, and the specific binding between the sample and NP-binding agent. The amount of specific binding between the sample and the NS1- (or NP-) binding agent can be indicative of the amount of NS1 (or NP) present in the sample. The presence of a significant amount of NS1 (or NP, or both) in the sample indicates that the subject is infected with influenza virus. Significance can be measured from an increase (with due allowance for experimental error) relative to a suitable control lacking the analyte(s) being measured.

The NS1-binding agent and the NP-binding agent can be contacted with the sample in contact with one another or separately. In the former situation for a solid phase assay, both binding agents can be deposited in the same area of a support. The agents can be deposited from separate solutions or together as mixture. Alternatively, for a liquid assay, the binding agents can be provided as a mixture in solution.

In assays in which the binding agent are kept separate, they can be deposited in different regions of the same support, such as in an array, or on different supports, such as beads. The separated binding agents can be contacted with the sample together (e.g., in an array format) or in separate assays. If separate assays are performed, a sample can be split or two samples can be obtained from the same subject.

Regardless of format, both agents can be contacted with a sample at substantially the same time (e.g., within the same hour, optionally within the same minute).

The measured amount of NS1 or NP in the sample can be compared to the amount of NS1 or NP in a control sample. The control sample can be a negative control lacking the analyte(s) being measured, optionally taken from a subject that is known or believed to be free from an influenza virus infection, e.g., is not showing at least one symptom of infection. As well as or instead of a negative control, comparison can be performed with a positive control, e.g., from a subject known or believed to have an influenza infection. Use of one or more positive controls from different times of infection can be used. For example the control sample can be known or believed to have been taken before 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days of infection, or optionally before 2 or 3 weeks of infection. Also for example, the control sample can be known or believed to have been taken at least after 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days of infection, or after 2 or 3 weeks of infection. Any combination of such parameters can be used. For example, the control sample can be known or believed to have been taken after at least 1 day and before 3 days; or after 3 days and before 3 weeks of infection. Also for example, the control sample can be known or believed to have been taken after at least 4 days of infection and before 2 weeks. Optionally, the control sample is taken from the same subject, but at a different timepoint (e.g., before, or within 1 or 2 days after, or at least 5 days after, the time at which infection is believed to have begun). Optionally, the control sample contains a known amount of NS1 and/or NP.

Lateral flow provides one convenient format for contacting the control and/or test sample with a binding agent. Such a format uses a detection agent and/or a capture agent. Usually, the detection agent is labeled and in solution and the capture agent is immobilized or immobilizable.

In another format, the test and control samples are immobilized and the samples are contacted with a binding agent in solution form, e.g., a solution of detection agent and/or capture agent. Optionally, the control sample can be contacted with the same solution of binding agent that the test sample is contacted with. For example, any viral proteins in the control sample and test sample can be immobilized onto solid support—for example, the control and test samples can be attached to separate beads or separate areas of a flat surface (e.g., in an array). The optionally-immobilized test and control samples can then be contacted with the same solution of the binding agent.

The test and control sample can be regarded as being contacted with the “same solution” of reagents (e.g., binding agents or SGC substrates) when the samples are contacted with a continuous body of reagent solution in a single vessel, or when the samples are contacted with divided aliquots of the reagent solution in separate vessel irrespective of whether the divided aliquots have been subjected to different processing between collection and analysis. For example, an aliquot of solution can be deposited onto (or into) a solid support or vessel (e.g., onto a membrane, for example as an array, or into a well of a multiwell plate) and evaporated to dryness, before the solid support is contacted to the sample, yet the test and control samples can be regarded as being contacted with the same “solution” of binding agent.

Binding agents can be regarded as being contacted with the same sample, if the binding agents are contacted with a continuous body of sample in a single vessel, or if the binding agents are contacted with divided aliquots of a sample in separate vessel irrespective whether the divided aliquots have been subject to different processing between collection and analysis, or if the binding agents are contacted with separate samples from the same individual collected at about the same time (i.e., within an hour of one another) again irrespective of whether the different samples have been separately processed between collection and analysis.

Contacting the test and/or control sample with the same solution of binding agent allows standardization of results to enable more reliable comparison of the amounts of NS1 and/or NP (e.g., the NS1:NP ratio) in test and control samples. When the sample is contacted with two or more binding agents (e.g., NS1-binding agent and NP-binding agent), the test and control samples are both optionally contacted with the same solution of the first binding agent and the same solution of the second binding agent. For example, the test and control can be contacted with a single co-solution of the first and second binding agents. Optionally, the test and control samples are also exposed to the same solutions of reagents and processed in substantially identical fashion. Optionally, the test and control samples are processed simultaneously.

The assessment of infection can include identifying one or more infected subjects in a population, and/or determining whether a particular subject is infected with influenza virus. Because the claimed methods comprise determining amounts of two “independent” influenza virus antigens (NS1 and NP), each of which individually are present in widely varying amounts within infected subjects, the overall sensitivity and reliability of the assay is increased. A combined NS1 and NP test is also less susceptible to false negatives by antigen mutations. In addition, in assays where a cosolution or mixture of detection agents for NS1 and/or NP is used, the combined signal generated by both reagents in the mixture can be stronger than either one alone. For example, antibodies to NS1 and NP can each be deposited onto two separate areas (e.g., as “stripes” in a lateral-flow assay) such that neither antibody is in contact with each other. Also for example, both antibodies can be deposited onto a single area or overlapping areas, e.g., as a single stripe. When both NS1 and NP antibodies are deposited together onto the same area, the binding of both NS1 and NP present in the sample to that area can generate a stronger signal than binding of NS1 alone or NP alone. Where both NS1 and NP capture agents are deposited in two separate areas, then infected samples in which one antigen is present in detectable amounts but the other antigen is not can also be detected.

As well as or instead of determining whether a subject is infected or not, the present invention provides methods that further characterize the nature of the infection (if present). The present inventors have discovered that the relative amounts of NS1 and NP vary during the progression of an infection. An increased or relatively higher NS1:NP ratio indicates an early stage of infection. Early stage of infection means that the infection began approximately less than one week ago, for example within the last three days, e.g., within the last 48 hours, optionally within the last 24 hours. Optionally, detection of NS1 indicates that the infection began at least about 6 hours ago. NS1 antigen appears early after infection relative to NP. The timing of antigen expression (during viral infection) of the two viral antigens appears to differ: NS1 is expressed very early, whereas NP is expressed later, although there is some overlap in expression.

Accordingly, one can monitor the course of infection and/or determine the stage of the infection by comparing the amounts of NS1 and NP in an infected subject. This information can be put to a wide variety of uses, including determination of the prognosis of an infected subject. Many anti-influenza treatments (e.g., TAMIFLU and Relenza) are more effective at an earlier stage of infection when viral reproduction is intensive (e.g., within 48 hours of infection), so that a higher NS1:NP ratio indicates a greater likelihood that such a treatment will be effective (e.g., in inhibiting the replication or propagation of virus). Conversely, a decreased or relatively lower NS1:NP ratio indicates a late or advanced stage of infection, with a lower chance that such drugs will be effective. Optionally, subjects who are found to be at a later stage of infection can be treated with agents known to have a therapeutic effect at the later stages of infection. Such treatments can include anti-inflammatories and regulators of the immune system, or inhibitors of airway congestion such as disodium cromoglycate. Antioxidants such as superoxide dismutase can also be effective in treating lung edema at late stages of infection.

The present method can be useful in a variety of ways. One can identify subjects at an early stage of infection from a population of infected subjects. Optionally, subjects in early stages of infection can be selectively administered a drug or test agent of interest. Such subjects with an early stage of infection can optionally be segregated or quarantined from a population of subjects, e.g., showing no symptoms of influenza infection. Asymptomatic or symptomatic subjects can also be tested for infection. One can also determine the variation in the efficacy of a treatment of interest at different stages of infection in order to determine an optimal time of administration for the treatment. Similarly, one can assess the efficacy of an anti-influenza drug by comparing the change in NS1:NP ratio over time in treated and untreated subjects. One can also screen one or more test agents for therapeutic efficacy against influenza.

Optionally, the detection assay is a non-PCR assay. A non-PCR assay is one that does not comprise the exponential amplification of a nucleic acid, for example by PCR (polymerase chain reaction) or other procedures. Accordingly, assays such as RT-PCR and immuno-PCR are considered to be PCR assays.

II. Influenza Viruses and their Proteins

The influenza viruses belong to the Orthomyxoviridae family, and are classified into types A, B, and C based upon antigenic differences in their nucleoprotein (NP) and matrix protein (M1). Further subtyping into subtypes and even strains is commonly based upon assessing the type of antigen present in two virion glycoproteins, namely, hemagglutinin (HA; H) and neuraminidase (NA; N). HA and NP are virulence factors mediating attachment of the virion to the surface of host cells. Thus, H5N1, H1N1 and H3N2 are examples of subtypes of influenza A that are of interest. Within each subtype there are hundreds of strains. M1 protein is thought to function in virus assembly and budding, whereas NP functions in RNA replication and transcription. In addition to these virion proteins, two other non-structural, i.e., non-virion, proteins are expressed in virus infected cells which are referred to as non-structural proteins 1 and 2 (NS1; NS2). The non-structural viral protein NS1 has multiple functions including the regulation of splicing and nuclear export of cellular mRNAs and stimulation of translation, as well as the counteracting of host interferon ability.

Optionally, the detection assay involves the detection of an extracellular fraction of the target analyte. For example, the detection assay detects NS1 protein that is present outside cells (e.g., due to the lysis of infected cells). Optionally, the detection assay can comprise or exclude conditions in which any cells present in the sample undergo a significant level of lysis or breaking open.

A. NS1 Protein

Commonly owned patent publications WO2008/048276 and US2007/0161078 set out the general concept that NS1 protein of influenza protein is an abundant protein in subjects infected with influenza viruses and thus useful for detection of these viruses. The NS1 is optionally detected using an anti-NS1 antibody. US2007/0161078 also shows, that the NS1 proteins of influenza A contain PL regions which can be readily detected using PDZ domains.

The NS1 protein has been identified and sequenced in influenza viruses and exemplary sequences can be found in the NCBI database. The NS1 proteins from influenza A, B and C do not in general show antigenic cross reactivity. Within a type (e.g., influenza A), there is considerable variation in sequence between subtypes, but some antigenic crossreactivity depending on which antibody is used. The GenBank accession numbers of some exemplary NS1 sequences from influenza type A, subtypes H1N1, H3N2 and H5N1 respectively, are CY003340 (SEQ ID NO:1003 AND SEQ ID NO:1004), CY003324 (SEQ ID NO:1005 AND SEQ ID NO:1006), and DQ266101 (SEQ ID NO:1007 AND SEQ ID NO:1008). The GenBank accession numbers of some exemplary NS1 sequences from influenza type B are AAA43690 (SEQ ID NO:1009 AND SEQ ID NO:1010) and BAD29872 (SEQ ID NO:1011 AND SEQ ID NO:1012). The NS1 protein in other strains of influenza either influenza type A, type B or type C, means a protein having the greatest sequence similarity to one of the proteins identified as an NS1 protein in known influenza strains of the same subtype, using as sequence for example, one of the GenBank accession numbers given above.

B. NP Protein

The NP protein has been identified and sequenced in many strains of influenza viruses and exemplary sequences can be found in the NCBI database. The GenBank accession numbers of some exemplary NP sequences from influenza type A for subtype H1N1 are NP 040982 (AAA43467) (SEQ ID NO:1013 AND SEQ ID NO:1014), for subtype H3N2 are AAZ38620 (YP308843) (SEQ ID NO:1015 AND SEQ ID NO:1016); and for subtype H5N1 are AY856864 (SEQ ID NO:1017 AND SEQ ID NO:1018) and AAF02400 (SEQ ID NO:1019 AND SEQ ID NO:1020). The GenBank accession numbers of some exemplary NP sequences from influenza type B are CAA32437 and ABF21293.

C. HA Protein

The HA protein has been identified and sequenced in many strains of influenza viruses and exemplary sequences can be found in the NCBI database. The GenBank accession numbers of some exemplary HA sequences from influenza type A for subtype H1N1 are AAB29091 and ABD59849, for subtype H3N2 are YP_(—)308839 and AAZ38616; and for subtype H5N1 are AAW72226. The GenBank accession numbers of some exemplary HA sequences from influenza type B are BAA96844.

D. M1 Protein

The M1 protein has been identified and sequenced in many strains of influenza viruses and exemplary sequences can be found in the NCBI database. The GenBank accession numbers of some exemplary M1 sequences from influenza type A for subtype H1N1 are NP_(—)040978.1, for subtype H3N2 are YP_(—)308841.1, or AAZ38617.1; and for subtype H5N1 are AAG48228 and AAO52905. The GenBank accession numbers of some exemplary M1 sequences from influenza type B are NP_(—)056664.1.

E. NA Protein

The NA protein, generally associated with late phase of infection, has been identified and sequenced in many strains of influenza viruses and exemplary sequences can be found in the NCBI database. The GenBank accession numbers of some exemplary NA sequences from influenza type A for subtype H1N1 are ABD59870, for subtype H3N2 are ABD59878; and for subtype H5N1 are ABF93438 and AAW72227. The GenBank accession numbers of some exemplary NA sequences from influenza type B are AAB26739.

III. Characterization of Infection, e.g., Combination with Other Assays

The determination of the NS1:NP ratio can be done using any method that helps to characterize the infection further. Where desired, subtype-specific agents can be used, which bind to a specific subtype of influenza virus but not others. The use of one or more of subtype-specific agents can allow simultaneous diagnosis, subtyping and/or prognosis of infection. Any combinations of pan-specific, type-specific, subtype-specific and/or strain-specific detection agents can be used. For example, PDZ proteins can be used to not only detect influenza antigens but also to distinguish between pathogenic and seasonal strains of influenza as well (discussed below).

Similarly, the NS1:NP ratio can be determined in conjunction with any other test or assay that can provide useful information about the infection. For example, assays for other influenza antigens can provide useful information about an influenza infection. The HA and M antigens are useful in typing influenza virus, while the M1 and NP antigens are used for subtyping and identifying strains of influenza virus.

The amount of influenza antigens, such as HA, MA, NS1 and/or NP can be determined by a variety of ways. The amount of genomic RNA or mRNA of influenza virus antigens can be assessed for example by hybridization or amplification (e.g., RT-PCR). The amounts of influenza antigens can be determined by contacting the sample with one or more agents that bind specifically to the antigen and detecting specific binding, for example through the formation of a complex between the antigen and one or more agents that bind specifically to the antigen. As discussed below, binding agents include antibodies and/or PDZ proteins. Any combination of agents can be used. If the sample is contacted with two binding agents specific for two (or more) different strains or subtypes of influenza virus, the different strains or subtypes can be identified by the relative binding of the binding agents. For example, if one binding agent is specific for a protein of influenza A and another for influenza B, then higher binding of the agent specific for influenza A than the agent for influenza B indicates that the influenza virus infection is influenza A. Reference to the relative binding includes situations in which the binding of one of two agents being compared is not detectable above background.

1. Detection and/or Typing of Influenza with PDZ Proteins

As discussed herein, multiple PDZ proteins bind to influenza proteins including NS1 and NP. Such PDZ proteins can be used in addition to or in lieu of antibodies, to determine the amounts of influenza antigens such as HA, MA, NS1 and/or NP in a sample. Any combination of PDZ polypeptides, PDZ polypeptides and/or antibodies that bind to antigens such as NS1 and/or NP can be used. A preferred format uses one or more PDZ domain as a capture reagent and one or more pan-specific antibodies as the detection reagent, although the reverse strategy can also be used. Table 1 indicates various PDZ proteins that can be used to detect influenza antigens.

TABLE 1 PDZ-PL Interactions RICI (SEQ ID NO: 13), NICI (SEQ NOS1 (PDZ # 1, 2, 3); MINT1 ID NO: 11), TICI (PDZ # 2); ZO-1 (PDZ #2); influenza A HA 8486126 (SEQ ID NO: 12) NSP; RIM2 NS1 8486133 ESEV (SEQ ID NeDLG (PDZ #1, 2); LIM-RIL;  NO: 2), RSEV (SEQ Vartul (PDZ #1, 2); MAGI2; ID NO: 7), RSKV DLG2 (PDZ #1, 2); MAST2; (SEQ ID NO: 8) DLG1 (PDZ # 1,2); PSD95 (PDZ # 1,2,3); MAGI1; TIP1; MAGI 3; Outer membrane protein; MAST2; Syntrophin gamma 1; MUPP1 (PDZ #13); PTPL1 (PDZ #2); Syntrophin 1 alpha; ERBIN; KIAA1526; AIPC; LIM mystique; TIP43; TIP2 influenza B HA 8486153 SICL (SEQ ID NOS1 (PDZ # 1, 2, 3); MINT1 NO: 18) (PDZ # 2); ZO-1 (PDZ #2); NSP; RIM2; Novel serine protease; PICK1 NA 8486155 DMAL(SEQ ID ZO-1 (PDZ #2); RIM2; Novel NO: 14), DMTL(SEQ serine protease; MINT1 ID NO: 15), DIAL(SEQ ID NO: 16) M1 8486158 RKYL(SEQ ID ZO-1 (PDZ # 2) NO: 29), KKYL (SEQ RIM2 d1 ID NO: 30) NP 8486160 DLDY (SEQ ID ZO-1 (PDZ # 2) NO: 17) RIM2 d1; syntenin

PDZ proteins can also be used to type or subtype influenza virus. Thus the claimed methods can include a test for distinguishing between pathogenic and seasonal subtypes of influenza A using. A more detailed description of the specificity of PDZ proteins for different types and subtypes of influenza virus is provided herein. PDZ proteins that binds strongly to NS1 include DLG1d1,2, LIM mystique d1, DLG2 d3, Vartul d2, PSD95 d1, Magi3 d1, DLG1d2, PTN-3 d1, DLG2 d1, NeDLG1 d1,2, Magi2 d5, DLG2 d2, and PSD95 d3 CS Bound, Magi2 d1, DLG1 d1, RhoGap10, Outer membrane, Magi1 d4, Tip 43, Tip1 d1, PSD95 d1,2,3, Tip33 d1, and PSD95 d2.

Other PDZ proteins that can be of use in the instant assays are described in PCT/US06/26155, filed Jul. 3, 2006, PCT/US06/41748 filed Oct. 21, 2006, PCT/US08/01123 filed on Jan. 28, 2008, as well as U.S. application Ser. Nos. 11/698,798 filed Jan. 26, 2007; 11/481,411 filed Jul. 3, 2006, each of which is incorporated by reference in its entirety.

NS1 from influenza A can contain a PL sequence that is specifically bound by various PDZ proteins. Table 2 below lists the PL sequences in NS1 of influenza A subtypes H5N1, H1N1 and H3N2. H5N1 is the most clinically relevant subtype of pathogenic strains. H1N1 and H3N2 are the most clinically relevant subtypes of seasonal influenza A. The table also indicates whether various PDZ domains bind to the indicated PL (as detailed further in Table 3). The table can be used to select PDZ domains for differential detection of pathogenic and seasonal subtypes of influenza A. For example, a PSD95 domain is useful for detecting pathogenic subtypes of influenza A, and INADL domain 8 is useful for detecting seasonal subtypes of influenza A. The PSD95 domain can be any of PDZ domains 1, 2, and 3 of PSD95, or combinations thereof. A preferred detection reagent is a protein formed from three copies of domain 2 of PSD95 in a PSD95. That is, three tandem copies interspersed by segments of PSD95 flanking its PDZ domains. In such a protein two of the copies of domain 2 of PSD95 effectively replace natural domains 1 and 3 of PSD95. Another preferred detection reagent is a protein containing PDZ domains 1, 2 and 3 of PSD95.

TABLE 2 PSD95 Influenza PSD-95 D1, D2, INADL A subtypes PL D2 D3 d8 H5N1 ESEV (SEQ ID NO: 2) ++ ++ − H1N1 RSEV (SEQ ID NO: 7) + +− ++ H3N2 RSKV (SEQ ID NO: 8) − − ++

Assay conditions such as buffer and temperature can be used to modulate binding to favor detection of a particular strain or differentiation among the different strains. The symbols used in Table 2 mean as follows: ++ relatively strong binding, + detectable but relatively weak binding, +/− detectable but relatively weak binding or undetectable binding, − undetectable binding. Detectable binding means that the signal from binding is greater in a sample containing NS1 of the indicated subtype relative to a control lacking the NS1 of the indicated subtype to a significant extent taking into account random variation due to experimental error. Undetectable binding means that the signal from binding to a sample containing NS1 of the indicated subtype is within the margin of error from the signal in a control lacking NS1 of the indicated subtype.

TABLE 3 Pathogen Protein C-terminus PDZ Partners influenza NS1 ESEV (SEQ Outer Membrane; PSD95 (PDZ # 2); PSD95 (PDZ A ID NO: 2) #1, 2, 3); DLG1 (PDZ #1); DLG1 (PDZ #1, 2); DLG1 (PDZ #2); DLG2(PDZ #1); DLG2 (PDZ #2); Magi3 (PDZ #1); PTN3 (PDZ #1); MAST2 (PDZ #1); NeDLG (PDZ #1, 2); Shank1 d1; Shank2 d1; Shank3 d1; Syntrophin1 alpha; Syntrophin gamma 1; Magi1 (PDZ #1); Magi1 (PDZ #4); Tip1; PTPL1 (PDZ #1); Mint3 (PDZ #1); Lym Mystique (PDZ #1); DLG2 (PDZ #3); MUPP1 (PDZ #8); NeDLG (PDZ #1); DLG5 (PDZ #1); PSD95 (PDZ #1); NumBP (PDZ #3); LIMK1 (PDZ #1); KIAA0313; DLG1 (PDZ #2); Syntenin (PDZ #2); Pick1 NS1 EPEV (SEQ PSD95 (PDZ # 2) ID NO: 27) PSD95 (PDZ #1, 2, 3) NS1 ESEI (SEQ Outer Membrane; PSD95 (PDZ #2); PSD95 (PDZ ID NO: 3) #1, 2, 3); NeDLG (PDZ #1, 2); DLG2 (PDZ #2); MAST2; PTN3 (PDZ #1) NS1 ESKV (SEQ PSD95 (PDZ #2); PSD95 (PDZ #1, 2, 3); MAST2; Magi3 ID NO: 4) (PDZ #1); NeDLG (PDZ #1, 2); NumBP (PDZ #4)

PDZ proteins can optionally be used to detect or quantify NS1 from influenza. NS1 from influenza A contains C-terminal PL sequences such as ESEV (SEQ ID NO:2), RSEV (SEQ ID NO:7), RSKV (SEQ ID NO:8). NS1 has been observed to bind to PDZ proteins such as NeDLG (PDZ #1, 2); LIM-RIL; Vartul (PDZ #1,2); MAGI2; DLG2 (PDZ #1, 2); MAST2; DLG1 (PDZ #1,2); PSD95 (PDZ #1,2,3); MAGI1; TIP1; MAGI 3; Outer membrane protein; MAST2; Syntrophin gamma 1; MUPP1 (PDZ #13); PTPL1 (PDZ #2); Syntrophin 1 alpha; ERBIN; KIAA1526; AIPC; LIM mystique; TIP43; and TIP2.

Similarly, PDZs that bind to NP from influenza A and/or influenza B can also be useful. For example, the influenza B NP contains the PL motif DLDY, bound by ZO-1 (PDZ #2), RIM2 domain 1; and syntenin. Other PL motifs in NP of influenza include: ACL (A/Duck/Shantou/2143/00(H9N2)), DNA (A/Quail/Hong Kong/G1/97 (H9N2)); ESA (A/Chicken/Hong Kong/NT16/99(H9N2)); LIL (A/swine/Boxtel/144#15/97(H1N1)); DNA (A/swine/Italy/1509-6/97(H1N1)); ENA (A/Chicken/Hong Kong/KC12/99(H9N2)); DNA (A/Chicken/Hong Kong/739/94(H9N2)); FDI (A/China); ENA (A/Silkie Chicken/Hong Kong/SF43/99(H9N2)); ENA (A/Chicken/Hong Kong/FY20/99(H9N2)); DNA (A/Chicken/Guangdong/10/00(H9N2)); ENA (A/Chicken/Hong Kong/SF2/99(H9N2)); STL (A/Swine/Colorado/23619/99(H3N2)); SGA (A/Swine/NorthCarolina/16497/99(H3N2)); ESA (A/Pigeon/Hong Kong/FY6/99(H9N2)).

An exemplary strategy for subtyping influenza A uses a PDZ from PDS95 as shown in Table 2 in combination with an INADL PDZ domain 8. As a general rule, detectable binding of the PSD95 domain without binding of the INADL domain or significantly stronger (i.e., stronger beyond experimental error) binding of the PSD95 domain that that of the INADL domain is an indication that the influenza A subtype is H5N1 (pathogenic). Conversely, detectable binding of the INADL domain to the sample without detectable binding of the PSD95 domain to the sample or significantly stronger binding of the INADL domain to the sample than of the PSD95 to the sample is an indication that the sample contains an influenza A subtype H1N1 or H3N2 (both seasonal influenza). Detectable but weak binding of PSD95 domain 2 to the sample compared with undetectable binding distinguishes H1N1 from H3N2 as indicated in the table. Detectable but relatively weak binding of PSD95 domains 1, 2 and 3 to a sample compared with binding of INADL to the sample is also an indication that the subtype is H1N1.

The use of domain 2 or domains 1, 2 and 3 of PSD95 and/or domain 8 of INADL are preferred as PDZ domain subtyping reagents. Preferred panspecific antibodies for use with a PDZ capture reagent are a pan specific antibody F68 8E6 (or an antibody that competes therewith) or F68 4B2 (or an antibody that competes therewith) as the detection antibody. The same or different panspecific antibody can be used with different PDZ domains in the same assay.

2. Detection of Influenza A with Pan Specific Antibodies

The invention also provides methods of detecting influenza A or influenza B in a manner that does not necessarily distinguish between subtypes of influenza A but can distinguish between influenza A and influenza B (or C). For example, influenza A can be detected using at least two pan specific antibodies to the NS1 protein of influenza A binding to different epitopes. The two panspecific antibodies specifically bind to different epitopes defined numerically as described herein or can be selected from different competition groups. Detection is preferably performed using a sandwich or lateral flow format as described in more detail below. One preferred combination of antibodies for detecting influenza A is F64 3H3 (or antibody that competes therewith) as the capture antibody, and F80 3D5 (or an antibody that competes therewith) as the detection antibody. Another preferred combination is F68 4H9 (or an antibody that competes therewith) as the capture antibody and F68 8E6 (or an antibody that competes therewith) as the detection antibody.

Detecting of influenza A using two panspecific antibodies can be combined with differential detection of influenza A subtypes as described herein. Such an assay indicates both whether influenza A is present, and if so, whether a pathogenic or seasonal subtype is present. The non-subtype-specific and subtype-specific assays can be performed separately or combined. One suitable format for combining the assays is to attach subtype-specific PDZ sequences that bind to NS1 to a solid phase, for use in subtype-specific or non-subtype specific analysis. Binding of a PDZ domain to an NS1 protein in the sample can be detected using a panspecific detection antibody. The panspecific detection antibody used to detect binding of the PDZ domain to the NS1 protein can be the same or different as the panspecific antibody used for non-subtype specific analysis. Thus, in a preferred format, one or more subtype-specific PDZ polypeptides (such as a PSD95 domain and/or an INADL domain 8) and at least one panspecific capture antibody for influenza A are attached to different regions of a support, and a common panspecific detection antibody (binding to a different epitope than the panspecific capture antibody) is used to detect binding of each of the capture reagents to an influenza A NS1 protein if present in the sample.

3. Detection of Influenza B with Pan Specific Antibodies

Influenza B can be detected using first and second panspecific antibodies to the NS1 protein of influenza B in analogous fashion to the assays described for detecting the NS1 protein of influenza A, as described. Such methods are performed using at least two pan specific antibodies to the NS1 protein of influenza B binding to different epitopes. The two panspecific antibodies bind different epitopes defined numerically as described above or can be selected from different competition groups. Detection is preferably performed using a sandwich or lateral flow format as described in more detail below. A preferred combination of antibodies for detection of influenza B uses F89 6B5 (or an antibody that competes therewith) as the capture antibody and F94 3A1 (or an antibody that competes therewith) or F94 1F9 (or an antibody that competes therewith) as the detection or detection antibody. Competition of antibodies is determined by binding to an NS1 protein of influenza B.

4. Combined Detection of Influenza A and Influenza B

Any one or more of the assays described herein can effectively be combined to provide an assay capable of detecting influenza A (non-subtype specific), influenza B (non-subtype specific), influenza A (pathogenic subtype) and influenza A (seasonal subtype). The individual assays can be performed separately or together—for example, one or more individual assays can be combined into a single assay. One suitable format for combining the assays is to attach a panspecific capture antibody for the NS1 protein of influenza A, a panspecific capture antibody for the NS1 protein of influenza B, a PDZ domain for a PL of a pathogenic subtype of influenza A (e.g., a PSD95 domain as discussed above), and a PDZ domain for a PL of a seasonal subtype of influenza A (e.g., an INADL 8 domain) to a single support. The support is contacted with a sample from a subject and at least two panspecific detection antibodies. One detection antibody specifically binds to the NS1 protein of influenza A at an epitope different from the capture antibody to the NS1 protein of influenza A. The other detection antibody specifically binds to the NS1 protein of influenza B at an epitope different from the capture antibody to the NS1 protein of influenza B. The complexes that form indicate whether influenza A and/or B is present, and if influenza A is present whether the influenza A is pathogenic or seasonal.

IV. Binding Agents for Detection of Influenza Antigens

The binding agent can be pan-specific or subtype-specific or strain-specific. For example, the agent optionally binds influenza antigens such as NS1 or NP in a pan-specific manner to influenza A or influenza B or influenza C antigens. For example, a pan specific agent for influenza A specifically binds to NS1 or NP from at least 2, 3 or 5 or all or substantially all known strains of influenza A.

Although pan-specific antibodies are preferred for use in detecting the antigen, any binding agent with specific affinity for the influenza antigen can be used as an antibody surrogate. One especially useful type of binding agent for influenza antigens includes PDZ proteins, discussed below. Other agents includes peptides from randomized phage display libraries screened against antigen from influenza A or B, and aptamers (RNA or DNA molecules selected in vitro from vast populations of random sequence that recognize specific ligands by forming binding pockets). Optionally, a binding agent includes a combination of one or more binding agents described herein. Any combination of binding agents set forth below can be used in detection assays of the invention.

A. Antibodies

Antibodies are a useful type of NS1 and/or NP binding agent. Such antibodies can include antibodies, both intact and binding fragments thereof, such as Fabs and Fvs, which specifically bind to a NS1 and/or NP. Usually the antibody is a monoclonal antibody although polyclonal antibodies can also be used. Examples of antibodies that can be expressed include mouse antibodies, chimeric antibodies, humanized antibodies, veneered antibodies and human antibodies. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen fragment including separate heavy chains, light chains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes one or more immunoglobulin chains that are chemically conjugated to other agents, or expressed as fusion proteins with other proteins. The term “antibody” also includes bispecific antibody. Unless otherwise indicated the antibodies described in the present application are mouse antibodies produced from hybridomas.

The agent can be an antibody that specifically binds to NS1 and/or NP. Optionally, the antibody is panspecific for different strains of influenza type A or B. Optionally, the antibody is subtype-specific or monospecific for a single strain of influenza type A or B. Optionally, the contacting step comprises, contacting the patient sample with first and second agents that specifically bind to different epitopes of NS1 and/or NP from influenza virus type A or B, and the first agent is immobilized on a support, and the detecting step detects a sandwich in which the first and second agents are specifically bound to the NS1 and/or NP protein to indicate presence of the virus. Optionally, the first and second agents are first and second antibodies. Optionally, the first and/or second agent is a polyclonal antibody. Optionally, the first and/or second agent is pan-specific for different strains of influenza type A or B.

Although antibodies are preferred for use in detecting the influenza antigen, any binding agent with specific affinity for the influenza antigen can be used as an antibody surrogate. Surrogates includes peptides from randomized phage display libraries screened against the antigen from influenza A or B. Surrogates also include aptamers. Aptamers are RNA or DNA molecules selected in vitro from vast populations of random sequence that recognize specific ligands by forming binding pockets. Aptamers can bind to nucleic acids, proteins, and even entire organisms. Aptamers are different from antibodies, yet they mimic properties of antibodies in a variety of diagnostic formats. Thus, aptamers can be used as a surrogate for panspecific antibodies.

1. NS1 Antibodies

Monoclonal antibodies to NS1 of influenza A that can be used to determine the amount of NS1 are disclosed herein. Table 4A list various monoclonal antibodies to NS1 of influenza A, including subtype-specific antibodies and pan-specific antibodies, e.g., pan-specific antibodies for detection of influenza, particularly influenza A. A pan specific antibody for influenza A specifically binds to the NS1 protein from at least 2, 3 or 5 or all or substantially all known strains of influenza A. Likewise a pan specific antibody for influenza B specifically binds to the NS1 protein from at least 2, 3, 5 or all or substantially all known strains of influenza B.

TABLE 4A H1N1 H3N2 H5N1A D S¹ R L Y¹ D S² R L Y² D S³ F63 1C5 − − − − D − R − DDDD N/A 1F9 DD − − − DDD N/A 3C1 − − − − DDD N/A 3G1 DD − D − R − DDD N/A 5E11 − − − − DDD N/A 2C3 − − − − DDD N/A F64 1A10 DDDD − RRR LLL DDDD − RRR LLL DDDD N/A 1D6 D S R L − DDDD − R − − − N/A 3H3⁴ DDD SSSS⁴ RRR LLL YY DD S RR − − DD N/A 5B4 D S R − − D − − − − − N/A 6C1 − S R − − − − − − − − N/A 6G12 D SSS RR LL − DD SS RRR LLL Y D N/A 7A8⁴ DD SSSS⁴ − − − − − RR − − − N/A 7H2 − − R − − − − − N/A 2H6 DD SSS − − − − − N/A 4C4 D SS R − − − DDD N/A 5H10 DD SSS R − − − D N/A 7D1 DDD SSS RRR − − − − − − − DDD N/A 8B3 D S RR LL − − − − L − − N/A 2H9 DD SSS − − − N/A 5G12⁴ DDDD SSSS⁴ RR L − − − − DDD N/A 7B1 − − − S − N/A 7B5 DDD S RRR LLL − DDDD − RR L − DDD N/A 8C11 − − − − − N/A 5G8 DDD tbd DDD − DDD N/A 6B6 − tbd DDD SSS DD N/A 6H1 DD tbd DD SS − N/A F68 4B2⁴ DD SSSS⁴ RRR LLL Y DDD SSSS RRR − Y − N/A 4H9 DDD SSS RRR LLL Y DDDD SSSS RR − Y DDD N/A 5B5 DDDD − RRR LLL − DDDD − RRR LLL − DDD N/A 6B7 DDDD S RRR LLL − DDDD S RRR LL − DDDD N/A 6D6 DD SSS RRR LLL Y DDD SSS RR − − − N/A 1D10 DDDD S RRR LLL − DDDD S RR LL − DDDD N/A 1E5 DDD SSS − − − N/A 3G5 DDD S − − − N/A 6A12 DDD S RRR LLL − DDD − RRR LL − DDD N/A 6C6 DD SSS R L DD S − − D N/A 7B10 DDD S RRR LLL − DDD − RRR LLL − DDD N/A 9A6 DD − − − − N/A 2C3 − − − − DDDD N/A 3H5 DD SS − − − N/A 4C1 DDDD SS RRR LLL − − − N/A 2H11 DDDD tbd DDD − DDDD N/A 4D6 DDDD tbd RRR LLL DDDD SSSS RRR LLL DDDD N/A 6A5 DD tbd RRR LLL DD S RRR LL D N/A 8A1 DDDD tbd RRR LLL DDDD − RRR LLL DDDD N/A 8E6 DDDD tbd RRR LLL DDDD SSSS RRR LLL DDD N/A 10A5 DDDD tbd RRR LLL DDDD SSSS RRR LLL DDDD N/A F70 1A3 DDD − R L DDDD − R L DDD N/A 2C4 DDDD S RRR LLL − DDDD S RRR LLL − DDDD N/A 2G11 DD − RR L DDDD − RR LL DDD N/A 1B2 − − D S − N/A 2D12 − S − − − DD SSSS RR LL − D N/A 2H1 DD SS RR L − DD SSSS R − − − N/A 3A6 D − DDD − − N/A 3C2 DD − RRR LLL DDD − RR LL DD N/A 3F6 DD − RR − DDD − RR − D N/A 3G7 − S − − − D SSS − − − − N/A 4G9 DD − RRR LLL DD − RRR LLL D N/A 4H10 DD − RR L DD − RR LL D N/A F72 1B11 DDD S RRR LLL − DDD S RRR LLL − DD N/A 1C1 DDD − RRR LLL DDD − RRR LLL DD N/A 1G4 − − − − DDD N/A 1H7 DD − RR LL DD − R LL D N/A 2A8 DD − RRR LLL DDD − RRR LLL DD N/A 3D7 DDD S − − DD N/A 1D9 DDDD tbd DDDD − DDDD N/A 2E7 DDDD tbd RRR LLL DDDD − RRR LLL DDDD N/A 2H7 DD tbd DDDD S DDDD N/A F80 3A9 − tbd DDD SSSS D N/A 3E7 DDD tbd RRR LLL DDD SSS RRR LLL DD N/A 4E7 DDD tbd RRR LLL DDD − RRR LL DD N/A 5E7 DDDD tbd DDDD − DDDD N/A 7E7 DDD tbd DD S DD N/A 7H4 DDDD tbd RRR LLL DDDD − RRR LLL DDDD N/A 3D5 DDD tbd RRR LLL DDD S RRR LLL DDD N/A 5B12 DD tbd DDDD − DDD N/A 6G12 DDDD tbd DDDD − DDDD N/A 7E8 DDD tbd RRR LLL DDDD S RRR LLL DDD N/A 8F6 DD tbd RR LL DDDD SSS RRR LLL DDD N/A 9B1 DD tbd RR LL DDDD SSS RRR LLL DDD N/A F81 1C12 D tbd R L DDD SSSS R L DD N/A 1F3 DDDD S RRR LLL DD S RRR LLL DDDD N/A 2B8 DD tbd − − DDD N/A 4D5 DDD tbd RRR ? DDD − RRR ? DDD N/A H5N1A H5N1B¹ H5N2 R L Y³ D S¹ R L Y¹ D F63 1C5 RRR LLL N/A − − − 1F9 N/A DDD − 3C1 N/A D − 3G1 RRR LLL N/A DDD − RRR LLL − 5E11 N/A − − 2C3 N/A − − F64 1A10 RRR LLL N/A DDDD − RRR LLL DDDD 1D6 − − N/A DD SSSS R − YY 3H3⁴ RR L N/A DDDD SSSS⁴ RR LLL YYYY DDDD 5B4 − − N/A DD SSS R − Y − 6C1 − − N/A D SSS − − − 6G12 RRR LLL N/A DDD SSSS RRR LL YYYY DDD 7A8⁴ − − N/A DDDD SSSS⁴ RRR LL YYYY DDD 7H2 N/A D S RR − − 2H6 N/A DD SSSS RR − 4C4 N/A DDD SSSS RRR LL 5H10 N/A DDD SSSS RRR LL 7D1 RRR − N/A DDDD SSSS RRR LLL YYYY 8B3 − LL N/A DD SSSS RRR LLL YYYY 2H9 N/A DD SSSS 5G12⁴ RRR LL N/A DDDD SSSS⁴ RR LLL 7B1 N/A D SSS 7B5 RRR LLL N/A DDD S RRR LLL − − 8C11 N/A DD SSSS 5G8 N/A DDD − 6B6 N/A DDDD SSSS DDD 6H1 N/A DDD SSSS DDD F68 4B2⁴ − − N/A D SSSS⁴ RR LL YYYY − 4H9 R L N/A DDDD SSSS R L YYYY DDDD 5B5 RRR LLL N/A DDDD − RRR LLL − DDDD 6B7 RRR LLL N/A DDDD S RRR LLL − − 6D6 R L N/A DD SSSS R L YYYY DDDD 1D10 RRR LLL N/A DDDD S RRR LLL − − 1E5 N/A − − 3G5 N/A − SSSS 6A12 RRR LLL N/A DDD S RR LLL − DDD 6C6 − − N/A DDD SSSS − − DDD 7B10 RRR LLL N/A DDD S RRR LLL − D 9A6 N/A − − 2C3 N/A − − 3H5 N/A DDD SSSS 4C1 − − N/A − − − − 2H11 N/A DDDD − − 4D6 RRR LLL N/A DDDD SSSS RRR LLL DDD 6A5 R L N/A DDD SS RRR LLL DDD 8A1 RRR LLL N/A DDDD − RRR LLL DDDD 8E6 RR LLL N/A DDDD SSSS RRR LLL DDDD 10A5 RRR LLL N/A DDDD SSSS RRR LLL DDDD F70 1A3 R L N/A DDD − R L DDDD 2C4 RRR LLL N/A DDDD S RRR LLL − DDDD 2G11 RR LL N/A D − R L DDD 1B2 N/A − − 2D12 RR LL N/A DDD SSSS R L YY − 2H1 − − N/A DDD SSSS − − YY − 3A6 N/A D − 3C2 RRR LLL N/A DDD − RRR LLL − 3F6 RR − N/A D − − − DDDD 3G7 − − N/A DDD SSSS − − Y − 4G9 RRR LLL N/A DD − RRR LLL DDDD 4H10 RR LL N/A DD − − − DDDD F72 1B11 RRR LLL N/A DDD SS RRR LLL − − 1C1 RRR LLL N/A DDD − RR LLL DDDD 1G4 N/A D − DDD 1H7 R LLL N/A DD − − LL DDDD 2A8 RRR LLL N/A DD − R LLL DDDD 3D7 N/A DDD S − 1D9 N/A DDDD S − 2E7 RRR LLL N/A DDDD − RRR LLL DDDD 2H7 N/A DDDD SS − F80 3A9 N/A − − − 3E7 − − N/A DDD SSSS RR LL DDDD 4E7 RRR LLL N/A DDD − RR LLL DDDD 5E7 N/A/ DDDD − − 7E7 N/A DDD S − 7H4 RRR LLL N/A DDDD − RRR LLL DDDD 3D5 RRR LLL N/A DDDD SSSS RRR LLL DDDD 5B12 N/A DD − DD 6G12 N/A DDDD − D 7E8 RRR LLL N/A DDDD S RRR LLL DD 8F6 RRR LLL N/A DDDD SSSS RR LL DDD 9B1 RRR LLL N/A DDD SSSS RR LL DDD F81 1C12 R L N/A DD SSSS R L D 1F3 RRR LLL N/A DDDD − RRR LLL DD 2B8 N/A DDD − − 4D5 RRR ? N/A DDD S RRR ? D ¹H1N1 and H5N1B were captured by PSD95 d1,2,3 in S2 ²H3N2 was captured by INADL d8 in S2 ³H5N1A does not have a PL so will not work in S2 ⁴3H3 and 7A8 remain at OD = 4.0 on H5N1B until 80 ng/mL, but weaken to OD = 2.0 on H1N1 at 80 ng/mL D = direct ELISA titer with MBP-NS1 − = OD450 of 0.0 to 0.4 at 160 ng/mL D = OD450 of 0.4 to 0.8 at 160 ng/mL DD = OD450 of 0.8 to 1.2 at 160 ng/mL DDD = OD450 of 1.2 to 1.6 at 160 ng/mL DDDD = OD450 of 1.6 and up at 160 ng/mL S = S2 ELISA titer with MBP-NS1 − = OD450 of 0.0 to 0.5 at 160 ng/mL S = OD450 of 0.5 to 1.5 at 160 ng/mL SS = OD450 of 1.5 to 2.5 at 160 ng/mL SSS = OD450 of 2.5 to 3.5 at 160 ng/mL SSSS = OD450 of 3.5 and up at 160 ng/mL R = Western with GST-NS1 − = negative R = very weakly positive RR = positive RRR = strongly positive L = Western with HA-NS1 lysate − = negative L = very weakly positive LL = positive LLL = strongly positive Y = S2 ELISA with HA-NS1 lysate − = OD450 of 0.0 to 0.5 at 160 ng/mL Y = OD450 of 0.5 to 1.5 at 160 ng/mL YY = OD450 of 1.5 to 2.5 at 160 ng/mL YYY = OD450 of 2.5 to 3.5 at 160 ng/mL YYYY = OD450 of 3.5 and up at 160 ng/mL

Pan-specific antibodies can be defined by reference to either a numerically defined epitope or by a competition group defined by reference to an exemplary antibody. For influenza A, pan specific antibodies preferably specifically bind to an epitope within residues 8-21, 9-20, 29-38 or 45-49 of FIG. 1A (SEQ ID NO:998) or FIG. 2 (SEQ ID NO:1000). The X's in this sequence can be any amino acid but are preferably an amino acid occupying the corresponding position in an NS1 protein from a strain of influenza, and more preferably the consensus amino acid occupying the corresponding position from at least two or preferably all known strains of influenza A. A consensus sequence of influenza A is provided in FIG. 2 (SEQ ID NO:1000). Some pan-specific antibodies specifically bind to an epitope within residues 9-11 or 13-16 of FIG. 1A (SEQ ID NO:998).

Pan specific antibodies can also be defined by a competition group; the antibodies within a competition group compete with one another for specific binding to the same antigen (i.e., an NS1 protein of influenza A or influenza B). Table 4B shows competition groups of panspecific antibodies binding to an NS1 protein of influenza A.

TABLE 4B Anti-Influenza A NS1 competition group mAb Ref. Group A F64 3H3 F68 4H9 Comment: Partial competition Group B F68 8E6 F80 3D5 Comment: Slight/ partial competition

Each group is defined by a prototypical antibody (in column 2) with which other antibodies (column 3) in the group compete. Groups A, B and C are preferred. All of these antibodies bind to the NS1 protein from at least strains H5N1, H1N1 and H3N2. The antibodies in different groups do not compete with each other.

Table 5 shows preferred antibodies for use in sandwich detection of the NS1 of H5N1 pathogenic strain of influenza A. In such assays, the detection agent is optionally an antibody preferably from Group A, or alternatively Group C or D. Optionally, the capture agent is a peptide comprising three copies of PSD95 domain 2 or PSD95 domains 1, 2 and 3.

TABLE 5 Anti-Influenza A H5N1 NS1 Mab or competition group PDZ Ref. Group A F68 4B2 F68 8E6 Group B PSD95(1, 2, 3) Group C F64 3H3

Table 6 shows competition groups for panspecific antibodies binding to the NS1 protein of influenza B.

TABLE 6 Anti-Influenza B NS1 competiton group mAb Ref. Group A: F89-1F4 F89 1F4 F89 F89 F89 F89 competitors 6D11 6G1 6H3 6B5 Group B: F94-3A1 F94 3A1 F94 competitors 7G2 Group C F94-1F8, F94-1F9 and F94-5E5 com- pete w/each other

Table 7 shows pairs of competing capture and detection antibodies. Detection antibodies are shown in the first row of the table and capture antibodies in the first column. Competition is shown with a C.

F89- F89- F89- F89- F89- F89- F89- F94- F94- F94- F94- F94- F94- 1F4 1G8 4D7 6B5 6D11 6G1 6H3 1F8 1F9 3A1 5E5 7A1 7G2 F89-1F4 C C C C C F89-1G8 C F89-4D7 C F89-6B5 C C F89-6D11 C C F89-6G1 C C F89-6H3 C C F94-1F8 C C C F94-1F9 C C C F94-3A1 C C F94-5E5 C C C F94-7A1 C F94-7G2 C C

Antibodies (including pan-specific antibodies) for influenza type B can also be described by epitope specificity with reference to the consensus sequence of NS1 proteins from influenza B strains shown in FIG. 2 (SEQ ID NO:1000). Preferred antibodies specifically bind to an epitope occurring within residues 10-28, 40-45, 50-57, 67-74, 84-100, 154-159, 169-173, 185-191, 212-224, 226-240 of FIG. 2, and particularly underlined regions thereof, which indicate residues that are invariable between different strains of influenza type B. Residues included in one of the above regions that are not underlined (i.e., vary between influenza type B strains) can be occupied by the consensus residue occupying that position shown in FIG. 2 or the residue occupying that position in any strain of influenza type B.

Some preferred combinations of antibodies to NS1 of influenza B for use in sandwich assays are indicated with a happy face in FIG. 13. For influenza A, the capture antibody is optionally F64 3H3, F68 4H9 and the corresponding detection antibody is F68 8E6 or F80 3D5. For influenza B, the capture antibodies is optionally F89 6B5 and the detection antibody is F94 1F9 or F94 3A 1. The detection antibody is optionally gold-conjugated. Antibodies reactive against seasonal and Avian Flu A NS1 can also be used to simultaneously determine pathogenicity.

Antibodies can be made from antigen-containing fragments of the protein by standard procedures according to the type of antibody (see, e.g., Kohler, et al., Nature, 256:495, (1975); and Harlow & Lane, Antibodies, A Laboratory Manual (C.S.H.P., NY, 1988) Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861; Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047 (each of which is incorporated by reference for all purposes).

Immunization can be biased to generate panspecific antibodies by immunizing with multiple strains of influenza A or B, or by immunizing with one strain and boosting with another. Alternatively, one can use a fragment from a highly conserved region of influenza A (e.g., 8-21, 9-20, 29-38 or 45-49 or at least three contiguous amino acids of any of these of SEQ ID NO:998) or B NS1 (e.g., 10-28, 40-45, 50-57, 67-74, 84-100, 154-159, 169-173, 185-191, 212-224, or 226-240 of SEQ ID NO:1001 or subfragments of at least three contiguous amino acids thereof) as the immunogen. Conversely, to generate a monospecific antibody, immunization with NS1 of a single strain, or a fragment of NS1 from a nonconserved region (e.g., a PL region of influenza A) is preferred.

2. NP Antibodies

A variety of monoclonal antibodies against influenza A and influenza B NP are known and/or commercially available. See, e.g., J. A. López, M. Guillen, A. Sanchez-Fauquier, and J. A. Melero, J. Virol. Methods 13:255-264, 1986 (describing 3 anti-NP monoclonal antibodies). The Examples used herein make use of the Capilia immuno-diagnostic assay from Taun, Inc. (Japan).

V. Detection Formats

The invention provides diagnostic capture and detect reagents useful in assay methods for identifying influenza A and/or B viruses in a variety of different types of biological samples. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, competitive and immunometric assays. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988); U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262;4,034,074, 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Contemplated formats include a lateral flow test (e.g., cassette or dipstick), flow through test, ELISA, western blot, and/or a bead-type array (e.g., wherein NS1 and NP are on different color beads).

Immunometric or sandwich assays are a preferred format (see U.S. Pat. Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375). A common type of “sandwich assay” optionally includes the use of a “capture” and a “detection” agent. Optionally, the capture and/or detection agent optionally comprises a combination of one or more binding agents described herein, e.g., an antibody, a PDZ, a population of antibodies and/or a population of PDZs. Optionally, a combination of capture agents for two or more different analytes is used. For example, the assays include capture agents for influenza NS1 and NP. Such assays optionally use one antibody or population of antibodies or a PDZ domain immobilized to a solid phase as a capture agent, and another antibody or population of antibodies or a PDZ domain in solution as detection agent. As discussed above, a combination of a capture PDZ domain and a detection antibody or vice versa is preferred for detection of influenza A. Typically, the detection agent is labeled (e.g., comprises a SGC that emits a detectable signal so that the presence and/or amount of the detection agent can be directly assessed without assessing the presence and/or amount of another detectable molecule). If an antibody population is used, the population typically contains antibodies binding to different epitope specificities within the target antigen. Accordingly, the same population can be used for both capture agent and detector agent. If monoclonal antibodies are used as detection and detection agents, first and second monoclonal antibodies having different binding specificities are used for the solid and solution phase.

Capture and detection agents can be contacted with target antigen in either order or simultaneously. If the capture agent is contacted first, the assay is referred to as being a forward assay. Conversely, if the detection agent is contacted first, the assay is referred to as being a reverse assay. If target is contacted with both capture agent and detection agent simultaneously, the assay is referred to as a simultaneous assay.

In the lateral flow format, the capture agent is optionally deposited (immobilized) on a solid support, e.g., a nitrocellulose membrane, onto a specified area—for example, shaped as a thin stripe, called a “test line.” Where multiple capture agents are involved (e.g., NS1 antibody and NP antibody), various capture agent formats can be used. In one such format, the NS1 and NP capture agents were each deposited onto separate areas (two-area format). In the second format, both NS1 and NP capture agents can be deposited onto the same area (the single-area format). An immuno-diagnostic assay detecting two analytes, e.g., the NS1 and NP antigens from influenza A (or B), can show superior results to a test detecting only one of the antigens (either NS1 or NP alone). For example, the results presented herein show that the signal strength for NS1 levels in some cases exceeded NP levels, and vice-versa. Thus, the dual-antigen test could detect influenzavirus samples with overall low viral load in more instances than a single-antigen assay for each antigen separately—e.g., where NS1 levels were detectable and NP levels were not, and vice versa. In addition, where both NS1 and NP levels were so low that neither generated a detectable signal within the detection limit of an assay for either antigen alone, the combined signal strength of both antigens in a single-area format sometimes resulted in a combined detection signal above the detection threshold. Finally, even in cases where NS1 and NP levels were both detectable, the dual antigen test had the additional advantage of allowing the determination of disease stage based on the NS1/NP ratio, which could be determined by comparing the signals generated from NS1 and NP alone (e.g., when using a two-are format, or by using two different labels for NS1 and NP (e.g., when using a single-area format).

When a lateral flow format is used, the results can be read at a predetermined timepoint after a sample is added to the lateral flow device. For example, the results can be read at 15 minute, 30 minutes, 1 hour, 2-8 hours, or 1-3 days.

After contacting the sample with capture and detection antibodies, a sample is incubated for a period that e.g., from about 10 min to about 24 hr, such as about 1 hr. A wash step can then be performed to remove components of the sample not specifically bound to the detection agent. When capture and detection agents are bound in separate steps, a wash can be performed after either or both binding steps. After washing, binding is quantified, for example by detecting SGC associated with the solid phase through binding of labeled detection agent (e.g., antibody) in solution. Usually for a given pair of capture and detection agents and given reaction conditions, a calibration curve is prepared from samples containing known concentrations of target antigen. Concentrations of antigen in samples being tested are then read by interpolation from the calibration curve. Analyte can be measured either from the amount of labeled detection agent in solution bound at equilibrium or by kinetic measurements of bound labeled detection agent in solution at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of target in a sample.

Competitive assays can also be used. In some methods, target antigen in a sample competes with exogenously supplied labeled target antigen for binding to an antibody or PDZ detection reagent. The amount of labeled target antigen bound to the detection reagent is inversely proportional to the amount of target antigen in the sample. The detection reagent can be immobilized to facilitate separation of the bound complex from the sample prior to detection (heterogeneous assays) or separation may be unnecessary as practiced in homogeneous assay formats. In other methods, the detection reagent is labeled. When the detection reagent is labeled, its binding sites compete for binding to the target antigen in the sample and an exogenously supplied form of the target antigen that can be, for example, the target antigen immobilized on a solid phase. Labeled detection reagent can also be used to detect antibodies in a sample that bind to the same target antigen as the labeled detection reagent in yet another competitive format. In each of the above formats, the detection reagent is present in limiting amounts roughly at the same concentration as the target that is being assayed.

Lateral flow devices are a preferred format. Similar to a home pregnancy test, lateral flow devices work by applying fluid to a test strip that has been treated with specific biologicals. Carried by the liquid sample, phosphors labeled with corresponding biologicals flow through the strip and can be captured as they pass into specific zones. The amount of phosphor signal found on the strip is proportional to the amount of the target analyte.

The lateral flow test can be designed by printing capture agents, for example a combination of anti-NS1 antibodies and anti-NP antibodies, onto a membrane. as either separate lines, or in combination in the same line.

A sample suspected of containing influenza is added to a lateral flow device, the sample is allowed to move by diffusion and a line or colored zone indicates the presence of influenza. The lateral flow typically contains a solid support (for example nitrocellulose membrane) that contains three specific areas: a sample addition area, a capture area containing one or more antibodies to NS1, and a read-out area that contains one or more zones, each zone containing one or more labels. The lateral flow can also include positive and negative controls. Thus, for example a lateral flow device can be used as follows: an influenza A and/or B NS1 protein is separated from other viral and cellular proteins in a biological sample by bringing an aliquot of the biological sample into contact with one end of a test strip, and then allowing the proteins to migrate on the test strip, e.g., by capillary action such as lateral flow. One or more antibodies, and/or aptamers are included as capture and/or detect reagents. Methods and devices for lateral flow separation, detection, and quantification are described by, e.g., U.S. Pat. Nos. 5,569,608; 6,297,020; and 6,403,383 incorporated herein by reference in their entirety. As an example, a test strip can comprise a proximal region for loading the sample (the sample-loading region) and a distal test region containing an antibody to an NS1 protein and buffer reagents and additives suitable for establishing binding interactions between the antibody any influenza B NS1 protein in the migrating biological sample. In another example, the test strip comprises two test regions that contain different antibodies to NS1 from two different subtypes of influenza B i.e., each is capable of specifically interacting with a different influenza B analyte.

The level of influenza B NS1 protein in a sample can be quantified and/or compared to controls. Suitable negative control samples are e.g. obtained from individuals known to be healthy, e.g., individuals known not to have an influenza viral infection. Specificity controls may be collected from individuals having known influenza A or influenza C infection, or individuals infected with viruses other than influenza. Control samples can be from individuals genetically related to the subject being tested, but can also be from genetically unrelated individuals. A suitable negative control sample can also be a sample collected from an individual at an earlier stage of infection, i.e., a time point earlier than the time point at which the test sample is taken. Recombinant NS1 of influenza B can be used as a positive control.

Western blots show that NS1 levels in biological samples are sufficient to allow detection of these antigens in a variety of different possible immunoassay formats. However, should the levels of NS1 in a particular biological sample prove to be limiting for detection in a particular immunoassay format, then, the live virus in a biological sample can be amplified by infecting cells in vitro, i.e., the NS1 protein in the virus-amplified sample should be detectable in about 6 hr to about 12 hr. The yield of NS1 antigen in biological samples and virus-amplified samples can also be improved by inclusion of protease inhibitors and proteasome inhibitors.

VI. Assay Sensitivity

As discussed above, the results of a test sample can be compared to that of a control sample to assess the amounts of NS1 and/or NP present. If so desired, the sensitivity of the detection assays of the invention can be assessed in comparison to the sensitivity of another “control” assay that is known to b sensitive enough to detect even very small amounts of the analyte (e.g., NS1 or NP) or entity of interest (e.g., influenzavirus). The control assay can for example be performed upon the same sample or organism in order to confirm the results of the detection assay. Alternatively, the control assay can be performed on different samples and/or different organisms the frequency of positive outcomes compared. The detection assay and control assay are optionally performed at the same timepoint—for example, both assays can be performed upon different samples taken from the same organism at the same timepoint. Optionally, the detection assay and control assay can be performed at different timepoints—for example, the control assay can be performed at a later timepoint, optionally after the organism displays at least one symptom of influenza.

Examples of useful control assays include isolation and cultivation in embryonated chicken eggs, identification of viral antigen in a commercial immunoassay test, immunodiffusion, hemagglutination and/or hemagglutination inhibition testing to identify the HA and/or NA subtype, RT-PCR detection of viral RNA or immunofluorescence detection of influenza A antigen in cells in respiratory specimens. A preferred control assay comprises RT-PCR for influenzaviral nucleic acid (e.g., genomic RNA within virions or mRNA within cells).

Positive predictive value, abbreviated PPV, can be measured as the percentage of samples that test positive in the instant methods and also test positive in a control assay for influenza virus. Optionally, PPV can be measured as the percentage of samples that test positive in the instant methods and also test positive in a control assay for an avian and/or pathogenic influenza virus. Preferably, the instant method has a PPV greater than about 20%, for example greater than about 40% or 60%, and most preferably greater than about 80%. Optionally, the assay is less sensitive than a PCR assay, for example the assay detects influenza in less than 95% of samples that test positive in an RT-PCR assay, or for example less than 90%, 80% or 70% of such samples.

Negative predictive value, abbreviated NPV, can be measured as the percentage of samples that test negative in both an assay of the invention and a control assay. Preferably, the instant method has an NPV greater than about 85% and most preferably greater than about 90%. The control assay used in determining positive or negative predictive value is usually an assay format among the most sensitive available formats, such as RT-PCR or immuno-PCR.

“True positive” means a sample or subject that is determined to be positive for influenza virus in a control assay. Similarly, “true positive avian influenza A” or “true positive highly pathogenic avian influenza A” indicates that the sample or subject was determined to be positive for avian or highly pathogenic influenza virus in a control assay. Conversely, “true negative” indicates a sample or subject that was determined to be negative for influenza virus in a control assay. Along the same lines, a “false positive” result indicates that the sample or subject was determined to be positive for the presence of an analyte or organism by an assay of the invention but was not determined to be positive in a control assay. Conversely, a “false negative” result optionally indicates that the sample or subject was determined to be negative for the presence of an analyte or organism in an assay of the invention, but was determined to be positive in a control assay.

“Background” can optionally be expressed as a percentage of false-positive or false-negative results.

Optionally, the detection limit of the assays of the present invention is above 0.1 pmoles, e.g., above about 1 pmole, sometimes above 10 pmole, for example about 100 pmoles, optionally above about 1 ng.

VII. Samples

Any biological sample from a subject can be used that contains or is thought might contain a detectable concentration of influenza proteins and preferably of NS1 and/or NP. For example, samples are often obtain from humans having or suspected of or at elevated risk of having influenza (e.g., through contact with others having influenza). Examples of samples that can be used are lung exudates, cell extracts (respiratory, epithelial lining nose), blood, mucous, and nasal swabs, for example. A high concentration of NS1 can be found in throat secretions and also in nasal and lung fluids. Thus, a preferred sample for identification of NS1 is a throat sample or a nasal sample. A nasal sample can includes materials such as fluids and/or tissues from the nasal cavity, the pharynx, nostrils and/or paranasal sinuses. Nasal fluid or tissue can be taken from the respiratory segment and/or the olfactory segment, such as conchae, the olfactory epithelium on the surface of the turbinates and the septum, and/or the vomeronasal organ, olfactory epithelium or olfactory mucosal cell types such as supporting (sustentacular) cells, basal cells, and Bowman's glands. Nasal samples can include lung fluids or tissues include those from the trachea, the primary bronchi and/or lungs, e.g., brochoalveolar fluid.

Binding of NS1 to an antibody occurs in the presence of up to 0.05% SDS, including 0.03% and 0.01%. Therefore, when the nasal, throat or other bodily secretion is not likely to easily be used in a lateral flow format, it can be treated with SDS. Preferably, the amount of SDS added is up to a final concentration of 0.01%, more preferably 0.03% and even more preferably, 0.05%.

VIII. Kits

Kits are provided for carrying out the present methods. The kits include one or more binding agents, typically antibodies or PDZ domains that specifically bind to NS1 of influenza A and/or B. The instant kit optionally contains one or more of the reagents, buffers or additive compositions or reagents disclosed in the examples. The kit can also include a means, such as a device or a system, for removing the influenza viral NS1 from other potential interfering substances in the biological sample. The instant kit can further include, if desired, one or more of various components useful in conducting an assay: e.g., one or more assay containers; one or more control or calibration reagents; one or more solid phase surfaces on which to conduct the assay; or, one or more buffers, additives or detection reagents or antibodies; one or more printed instructions detailing how to use the kit to detect influenza A and/or B, e.g. as package inserts and/or container labels, for indicating the quantities of the respective components that are to be used in performing the assay, as well as, guidelines for assessing the results of the assay. The instant kit can contain components useful for conducting a variety of different types of assay formats, including e.g. test strips, sandwich ELISA, Western blot assays, latex agglutination and the like.

IX. Antibody Arrays

The invention further provides arrays of antibodies and/or PDZ domains immobilized at different regions. Such arrays include a plurality of different antibodies and/or PDZ domains in different regions of the array, each with specificity for influenza proteins such as M1, HA, NA, NS1 or NP of influenza A and/or B. The different antibodies can be selected to have specificity for different subtypes and/or strains of influenza A and/or B. Antibodies that are panspecific for influenza A and/or B NS1 can also be included. Antibodies for influenza A or C NS1 proteins can also be included. Such arrays are useful for detection of influenza A and/or influenza B, and/or influenza C and distinguishing between subtypes and strains of these viruses.

Numerous formats for arrays have been proposed. U.S. Pat. No. 5,922,615 describes a device that utilizes multiple discrete zones of immobilized antibodies on membranes to detect multiple target antigens in an array. U.S. Pat. Nos. 5,458,852, 6,019,944, U.S. Pat. No. 6,143,576 and U.S. patent application Ser. No. 08/902,775 describe diagnostic devices with multiple discrete antibody zones immobilized in a device but not on a membrane for the assay of multiple target antigens. In U.S. Pat. No. 5,981,180, microspheres are again used to immobilize binders (including antibodies) and the microspheres are distinguished from one another without separating them from the sample by detecting the relative amounts of two different fluorophores that are contained in the microspheres in order to identify the specific binder attached to the microsphere.

All publications, and patent filings cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Genbank records referenced by Genbank identification (GID) or accession number, particularly any polypeptide sequence, polynucleotide sequences or annotation thereof, are incorporated by reference herein. If more than one version of a sequence has been associated with the same accession number at different times, reference to a deposit number should be construed as applying to the version in existence at the effective filing date of the application. Various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. For example, although the invention has been described primarily for influenza A and influenza B, a similar strategy can be used mutatis mutandis to detect influenza C. Unless otherwise apparent from the context, any feature, step or embodiment can be used in combination with any other feature, step or embodiment.

EXAMPLES Example 1 NS1 Protein is Expressed in Human Clinical Specimens

Human nasal secretions were examined for the presence and amount of NS1 from Influenza A. Human nasal aspirates were collected and stored in M4 viral transport media (Remel, Inc, Lenexa, Kans.) at −80° C. Stored material was thawed and run on 10% SDS-PAGE. Western blot analysis was performed with monoclonal antibodies to NS1, 3H3 and 1A10 (Arbor Vita Corporation, Sunnyvale, Calif.). The results for six samples are shown in FIG. 4. The results show that NS1 is present in large amounts in nasal secretions.

To investigate the timeline of when NS1 was produced and secreted by cells infected with influenza A virus, MDCK cells were infected with human influenza ProteinPR/8 at a MOI of 0.1. Supernatant as well as cells were collected and lysed in 1% Triton X-100 and subjected to SDS-PAGE and western analysis with monoclonal antibody 3H3 which is pan-reactive to NS1. NS1 was detected in infected cells within 24 hours after infection and detected in the supernatant of infected cells within 48 hours (see FIG. 5). This suggests that a NS1 based diagnostic may be able to detect infection by influenza A within 48 hours and possibly within 24 hours.

Example 2 NS1 Interacts with PDZ in Cells

To verify that NS1 interacts with PDZ proteins in cells, a series of PDZ pull-down experiments were performed. 293 HEK cells were transfected with plasmids containing HA-NS1-H5N1B or with HA-NS1-H3N2. Lysates were prepared as described herein. Glutathione-sepharose-PDZ beads were prepared (10 ug of DLG1d1,2, 10 ug of NeDLGd1,2, and 10 ug PSD95d1,2,3) and used to pulldown 150 ug of lysate from transfected 293ET cells as shown in FIGS. 6 and 7. Following an overnight incubation at 4° C. and multiple washes with HNTG buffer, a membrane was prepared with the pulldowns. The membrane was probed with F63-3G1 supernatant (1:5). All 3 of the PDZs tested successfully pulldown NS1 from cell expressing HA-H5N1B (see FIG. 6).

Similarly, glutathione-sepharose-PDZ beads were prepared (40 ug of INADLd8) and used to pulldown 150 ug of lysate from 293ET cells transfected with H3N2. Following an overnight incubation at 4° C. and multiple washes with PBS, a western blot was prepared and probed a-HA (1:500) (Roche). INADL d8 successfully pulldown HA-H3N2 NS1 from cell lysate (FIG. 7).

The conclusion is that the NS1 PL is functional within the cell and can interact with PDZ domains as determined by the MATRIX assay.

Example 3 Monoclonal Antibodies to NS1

Monoclonal antibodies were prepared to specifically bind to subtype NS1 proteins (e.g., H5N1), NS1 PL classes (e.g., ESEV (SEQ ID NO:2)) and for pan-specificity (influenza A). The strategy for the generation of monoclonal antibodies to NS1 is as follows:

-   1. GST and MBP fusion proteins of NS1 were generated. The cloning     vectors were obtained from Pharmacia (GST) or New England Biolabs     (MBP). The NS1 coding regions were synthesized using overlapping     oligonucleotides by DNA 2.0 (Menlo Park, Calif.). -   2. Mice were immunized with MBP-NS1 fusion proteins at doses ranging     from 10-100 ug per dose in CFA then IFA and PBS. -   3. Splenocytes and lymphocytes were harvested 3 days after the last     boost with the corresponding GST-NS1 fusion protein and fused with     FOX-NY myeloma cells according to Kohler and Milstein (Nature 1975). -   4. The hybridomas were screened first with MBP-NS1 in an ELISA. The     positive wells were cloned and rescreened with a panel of MBP and     GST NS1 and classified into pan-reactive or subtype reactive. -   5. Further screenings were done using Western blots to verify the     molecular weight of the target protein that is consistent with NS1. -   6. An additional screening was performed using a S2 assay format     (see Example 4) for compatibility with PDZ capture. -   7. Steps 5 and 6 were repeated with eukaryotic expressed NS1 in the     form of a cell lysate. -   8. The antibodies are checked for compatibility with a lateral flow     format described in Example 4.

Finally, the antibodies are checked for the ability to detect NS1 in a clinical specimen. FIG. 20 shows the binding profile of a wide variety of monoclonal antibodies to NS1 from various subtypes of influenza A.

Example 4 Detection of NS1 Using a Lateral Flow Format

Examples of lateral flow formats for detection of NS1 are provided in FIGS. 8, 9 and 10A-F. FIG. 8 provides a lateral flow using PDZ capture followed by monoclonal antibody detection. For all cases, recombinant PDZ domain proteins or antibodies were deposited on RF120 Millipore membrane using a striper. For FIG. 8, the PDZ proteins PSD95D1-3, and INADL D8 were deposited at a concentration of 0.5 mg/ml. A control band was also deposited composed of goat anti-mouse antibody (GAM) also at 0.5 mg/ml. NS1 protein was combined with gold conjugated monoclonal anti-NS1 such as 4B2 in 100 ul volume in TBS-T buffer. The NS1 proteins used were from H1N1, H3N2, H5N197, H5N1, and a control lane did not contain NS1. In all cases, human nasal aspirates were diluted and stored in saline or M4, as indicated. The samples were directly mixed with gold conjugated antibody in the amounts described below.

The PDZ striped membrane was inserted into the NS1/anti-NS1 protein solution and flow initiated by capillary action and a wicking pad. NS1 was subtyped based on the pattern of PDZ reactivity; H1N1 binds to both PSD95 and INADL d8; H3N2 binds to INADL d8 only; H5N1 binds to PSD95 only. Influenza A subtyping was performed based on the results of the NS1 lateral flow using reactivity to PDZ and detection with a gold conjugated pan-reactive anti-NS1 monoclonal antibody.

In FIG. 9, 13 different monoclonal antibodies were deposited on the lateral flow device. The 13 antibodies used were F64-1A0, F64-3H3, F64-6G12, F64-7A8, F64-7D1, F68-1D10, F68-4B2, F68-4H9, F68-6A12, F68-6B7, F68-6D6, F68-7B10. A subtype specific gold conjugated pan-NS1 antibody was added to a sample containing H1N1 influenza virus. The sample was applied to the lateral flow device and the results are shown in FIG. 9. The results show that a pan-specific antibody can be used for the test and the assay identified which antibodies were the best for binding to H1N1. The binding strength is quantified by using the following symbols: (−) for no binding, (+) for weak binding, (+++) for strong binding and (++) for moderate binding.

A lateral flow assay to identify pathogenic Influenza A in a patient sample is produced having pan-specific antibodies deposited on the membrane. The patient sample is admixed with a mixture of gold-labeled antibodies that recognize all NS1 PL's. The sample is applied to the lateral flow test strip and if a pathogenic strain of influenza A is present a line is formed on the strip.

The strip tests were run using the following protocol and materials: strips previously striped with goat anti-mouse/PSD95 d1,2,3/INADL d8; TBST/2% BSA/0.25% Tween 20 buffer; Stocks of NS1 proteins MBP-H1N1, MBP-H3N2, MBP-H5N1A, and MBP-H5N1B “old” (Jon's) fast gold-conjugated F68-4B2 antibody; and Maxisorp ELISA plates. The procedure was performed as follows:

-   1) Stock NS1 proteins were diluted down in TBST/2% BSA/0.25% Tween     20 to 100 ng/uL (using no less than 5 uL of proteins to perform the     dilutions) -   2.) The 100 ng/uL dilution was diluted down to 50 ng/uL by adding 10     uL of the protein to 1 0 uL of TBST/2% BSA/0.25% Tween 20 -   3.) A stock solution of gold-conjugated antibody in TBST/2%     BSA/0.25% Tween 20 buffer was prepared. Four uL of the antibody was     added to every 100 uL of the buffer, and enough buffer was prepared     for 6 100 uL reactions (which provides extra dead volume). -   4.) 98 uL of the antibody/buffer mix was added to separate wells in     the ELISA plate -   5.) 2 uL of the NS1 dilutions were added to the buffer-containing     wells (one NS1 per well) -   6.) One well was left with just antibody and buffer to serve as a     negative “no NS1” control -   7.) The ELISA plate was tapped several times to mix the contents of     the wells -   8.) The pre-striped strips were added to the wells and observed     during development.

After approximately 15 minutes (when all of the liquid had been absorbed, but the strip was not yet dry) the strips were removed from the wells and scanned into the computer.

The test provided in FIGS. 10A and 10B was prepared as follows: a GST-PSD95 d1,2,3 protein was striped onto the membrane at 3 mg/mL for the avian test, or alternatively a mixture of two monoclonal antibodies can be used (1.1 mg/mL F64-3H3 and 0.075 mg/mL F68-4H9 for the pan-flu A test. A second line of 1 mg/mL polyclonal goat anti-mouse antibody was used for the test capture line. The steps are set out below.

1. Prepare cards with a sample membrane and sink pad.

2. Stripe membrane with the PDZ protein and/or antibodies (see above for conc.)

3. Dry the membrane overnight at 56 degrees, then cut the cards into strips 4.26 mm wide.

4. Attach a glass fiber sample pad to the bottom of the strip and place the entire strip inside a cassette for testing.

5. Thaw sample to be tested and add 80 μl of sample to 20 μl of buffer. Pipette up and down several times to mix.

6. Spike 8 μl of the gold-conjugated (Au—) detector mix into the sample/buffer solution. This detector mix is 4 μl of Au-F68-4B2 with 4 μl of Au-F68-3D5. Pipette up and down several times to mix.

7. Add 100 μl of the prepared sample to the sample well on the cassette.

8. Read the test and control lines on the cassette at 15 minutes post-addition of sample. The control line is clearly visible for any test results to be read reliably. Flu A positive samples are noted with (+). Flu A negative samples are noted with (−). The top arrow is pointing to the control and the bottom arrow is pointing to the test.

In both cases the top line is a control line (goat anti-mouse mAb), the second line down is the test line (mixture of F64-3H3 and F68-4H9 mAbs for the Pan-Flu A Test and PSD95 d1,2,3 for the Avian test). 2 ng of H5N1 protein was tested for the Avian test. The bottom circular spot is the sample well. In FIG. 10 a, both test are positives.

FIG. 10C shows three of twenty human samples that were tested with the format shown in FIGS. 10A and 10B. The samples showed a variety of outcomes, for example, Sample 1 was positive for Flu A, but negative for Avian Flu A (i.e., H5N1) and Sample 14 was negative for both (i.e., FluA and H5N1). FIG. 10 d shows the same test for H1N1, H3N2, and H5N1 recombinant proteins. The Pan-FluA test was positive for all three. The Avian Flu test was positive for only H5N1.

In FIG. 10E, Gold-conjugated PDZs were used as detectors and single or multiple mAbs were used for capture. FIG. 10E had liquid gold added in the form of Au-PSD95 d1,2,3 with a F68-4B2 mAb capture. 1.7 ng of NS1 H5N1 protein tested positively. This was an Avian Flu (i.e., H5N1) specific test.

In FIG. 10F, a dried gold method was used. The cards were prepared as in the liquid gold protocol except the sample pad was affixed to the card before striping. When the captures were striped down, the gold-conjugated detector mix (which here also contained a conjugate diluent) was sprayed on the sample pad at the base of the card. The cards were dried, cut, and placed in cassettes as with the liquid test. When the human samples were prepared, they were treated with only the buffer solution before 100 μl was run on the cassette (no additional gold-conjugated detector mix was added). The Flu A positive samples are noted with a (+), the Flu A negative samples are noted with a (−). These cassettes were designed and read in the same way as the liquid gold cassettes. In FIG. 10F, Sample 7 and 9 were positive for both Flu A and Avian flu (i.e., H5N1) and sample 12 was negative for both Flu A and Avian flu (i.e., H5N1).

Example 5 Detection of Influenza B NS1 Using Panspecific Antibodies

Using anti-Influenza B NS1 monoclonal antibodies generated according to the above method, a lateral flow test was developed to detect Influenza B NS1. Monoclonal anti-influenza B NS1 antibodies were deposited on an HF075 Millipore membrane at a concentration of ˜0.7 mg/ml using a striper. Some examples of antibodies deposited as capture agents are among the following: F89 1F4, F94 3A1, F89 4D5. A control band was also deposited composed of goat anti-mouse antibody (GAM) also at 1 mg/ml. Flu B NS1 protein was combined with gold conjugated monoclonal anti-NS1 such as F94 3A1 (when F94 3A1 is not used as capture) in 100 μl volume of AVC Flu B buffer. The FluB NS1 proteins used were either recombinant AVC ID 522 (B/BA/78 NS1) and AVC ID 523 (B/YM/222/2002) or clinical samples of from patients known to be infected with influenza B.

The anti-Flu B NS1 antibody striped membrane was inserted into the FluB NS1/anti-NS1 protein solution and flow initiated by capillary action and a wicking pad.

Several combinations of anti-Flu B NS1 capture and detection agents were used in several experiments. The following is an example protocol. The strip tests were run using strips previously striped with goat anti-mouse/F89 1F4 anti-Flu B NS1 monoclonal antibody; 90% M4 viral transport media, 10% of a 10×AVC Flu B buffer; Stocks of NS1 proteins MBP-Flu B NS1 (AVC 522 and AVC 523); gold conjugated F94 3A1 antibody; and Maxisorp ELISA plates. The procedure was performed as follows:

-   1) Stock NS1 proteins were diluted down in 90% M4 viral transport     media, 10% of a 10×AVC Flu B buffer -   9.) The stock of NS1 was diluted down to 0.5 ng/μL by diluting with     90% M4/10% of a 10×AVC Flu B buffer. -   10.) Four μL of the gold-conjugated antibody was added to every 100     μL of the buffer -   11.) 98 μL of the antibody/buffer mix was added to separate wells in     the ELISA plate -   12.) 2 μL of the NS1 dilutions were added to the buffer-containing     wells (one NS1 per well) to achieve the desired final protein     concentration (example 1 ng Flu B NS1) -   13.) One well was left with just antibody and buffer to serve as a     negative “no NS1” control -   14.) The ELISA plate was tapped several times to mix the contents of     the wells -   15.) The pre-striped strips were added to the wells and observed     during development.

After approximately 15 minutes (when all of the liquid had been absorbed, but the strip was not yet dry) the strips were removed from the wells and scanned into the computer.

FIG. 11 shows results from testing various pairs of monoclonal antibodies as capture and detection reagent on two strains of influenza B, B/BA78 (also known as strain 522), and B/Yagamata\222\2002, also known as strain 523). The four different panels show four combinations of antibodies. In each panel, tracks 3 and 6 are negative controls. Tracks 1 and 2 are recombinant NS1 from strain 522 and tracks 4 and 5 are recombinant NS1 from strain 523. The presence of additional bands in tracks 4 and 5 but not tracks 1 and 2 of the first panel shows that the F89-F4 capture antibody F89-4G12 detection antibody combination detects the 523 strain but does not detect the 522 strains. The other panels can be analyzed in the same way. The results from this experiment and other similar experiments are summarized in FIG. 13. FIG. 13 shows which antibodies can serve as a capture antibody and which as a detection antibody and whether the antibodies are panspecific for both strains of influenza B (522 and 523) or monospecific to 522 or 523. For example, the F89-1F4 antibody can serve as either a capture or detection antibody and is panspecific. F94-4C10 works as a detection antibody but not as a capture antibody and is specific for influenza B 523. F89-1F4 and F94-3A1 are preferred antibodies for use in lateral flow format.

A lateral flow assay was used to identify Influenza B in a patient sample is produced having pan-specific antibodies deposited on the membrane. The patient sample was admixed with a mixture of gold-labeled antibodies that recognize all Influenza B NS1s. The sample was applied to the lateral flow test strip. Presence of influenza B is present a line is shown by a line formed on the strip. FIG. 12 shows the results from different dilutions of a patient sample compared with positive and negative controls. The upper part of the figure shows the actual appearance of lines indicating presence of influenza B. The lower part of the figure indicates the relative intensity of the bands. Influenza B was easily detectable up to a dilution of at least 400 fold.

Example 6 Detection of Influenza A in Clinical Samples Using Antibody-Based Assays for Both NS1 and NP

A lateral flow assay was devised as described in Example 4, using a capture antibody that was pan-specific for influenza A NS1 and a second detection antibody that was similarly pan-specific for influenza A NS1 (hereafter called the “AVC Flu A/B” test). To assess the relative sensitivity of the AVC Flu A/B test, three human clinical samples collected in BD viral transport media were diluted down to the limit of detection (LOD) of the AVC Flu A/B test. Specifically, sample 5409 was diluted 2-fold, sample 5415 was diluted 800-fold and sample 5111 was diluted 40-fold. The diluted samples were then tested for the presence and/or levels of NS1 by the AVC Flu A/B test. The same samples were also tested for the presence of the NP antigen of influenza A by a commercially available rapid immunodiagnostic assay (Capilia™ Flu test from Tauns, Inc., Japan—hereafter, the “Tauns test”) that uses an antibody for NP from influenza A. The results were read by an electronic reader (CAMAG) after about 6, 15 and 30 minutes. The visible threshold was determined to be 15 AU units. Samples were considered positive if the test readout is >15 AU units. If variations >30% between measurements, the data was discarded. FIG. 15 compares the results of the two assays. The AVC Flu A/B test detected NS1 in 2 of 3 diluted clinical specimens at the 15-minute timepoint, and 3 of 3 samples at 30 minutes. The Tauns test detected NP in only one of the three clinical specimens in 15 minutes, and in two of three samples at 30 minutes.

Similar results were also obtained for three new clinical samples. The samples were diluted in BD viral transport medium. Sample 5248 was diluted 80-fold, sample 5249 was diluted 20-fold, and sample 5312 was diluted 80-fold. The final buffer composition used in the three tests were as follows. For the Tauns test (NP, FLU7H101), the final buffer composition was 50% sample in BD VTM and 50% BL Tauns liquid extraction buffer. For the AVC NP test, the final buffer composition was 50% sample in BD VTM and 50% AVC Buffer 1. For the AVC Flu A/B test (NS1) the final buffer composition was 50% sample in BD VTM and 50% AVC Buffer 1. The tests were allowed to develop for about 6, 15 and 30 minutes. As shown in FIG. 17, the AVC Flu A/B detected NS1 in all 3 clinical specimens in 15 minutes, while the Tauns test failed to detect NP in any of the three samples.

To confirm these results, eight new clinical samples were similarly diluted and tested. As shown in FIG. 16, the AVC Flu A/B test detected NS1 in 8 out of 8 clinical specimens in 15 minutes. The Tauns test detected NP in 3 out of the 8 samples in 15 minutes. The results at 15 and 30 minutes are summarized in Tables 8A and 8B below.

TABLE 8A Comparison of influenza A test results at 15 minutes (highly diluted samples) Sample Dilution Sample Tauns NP AVC NS1 ID factor Type* Test Test 5325 1/800 A-Positive − + 5336 1/80 A-Positive − + 5589 1/25 A-Positive − + 5901 1/20 A-Positive − + 5415 1/10 A-Positive + + 5249 ½ A-Positive − + 5248 ⅕ A-Positive + + 5632 ½ A-Positive + + Neg. Control Negative − − Concordance with PCR: 38% 100% *A-Positive by PCR

TABLE 8B Comparison of influenza A test results at 30 minutes (highly diluted samples) Sample Dilution Sample Tauns NP AVC NS1 ID factor Status* Test Test 5325 1/800 A-Positive − + 5336 1/80 A-Positive − + 5589 1/25 A-Positive + + 5901 1/20 A-Positive + + 5415 1/10 A-Positive + + 5249 ½ A-Positive + + 5248 ⅕ A-Positive + + 5632 ½ A-Positive + + Neg. Control Negative − − Concordance with PCR for Flu A+: 75% 100% *A-Positive by PCR

Tables 8C and 8D summarize results at high concentrations.

TABLE 8C Comparison of influenza A test results at 15 minutes (concentrated samples) Sample Dilution Sample Tauns NP AVC NS1 ID factor Status* Test Test 5409 ½ A-Positive − − 5111 ″ A-Positive + + 5581 ″ A-Positive − − 5634 ″ A-Positive − + 5349 ″ A-Positive − + 5411 ″ A-Positive − + 5312 ⅕ A-Positive + + 5336 ″ A-Positive + + 5589 ″ A-Positive + + Neg. Control Negative − − Concordance with PCR for Flu A+: 44% 77% *A-Positive by PCR

TABLE 8D Comparison of influenza A test results at 30 minutes (concentrated samples): Sample Dilution Sample Tauns NP AVC NS1 ID factor Status* Test Test 5409 ½ A-Positive − + 5111 ″ A-Positive + + 5581 ″ A-Positive − + 5634 ″ A-Positive − + 5349 ″ A-Positive − + 5411 ″ A-Positive + + 5312 ⅕ A-Positive + + 5336 ″ A-Positive + + 5589 ″ A-Positive + + Neg. Control Negative − − Concordance with PCR for Flu A+: 55% 100% *A-Positive by PCR

Tables 8E and 8F give a more quantitative summary of signal strengths observed for more concentrated samples. At 15 minutes, 6 of 8 total samples tested positive using the AVC Flu A/B test, while 2 of the 8 samples tested positive using the Tauns test. At 30 minutes, all 8 samples tested positive using the AVC Flu A/B test, while 4 of the 8 samples tested positive using the Tauns test.

TABLE 8E Sensitivity of both diagnostic tests with less diluted samples 15′ 30′ sample# final % tauns AVC tauns AVC 5409 50% 9 9.5 8.4 16.4 50% 5.3 11.5 6.5 23.1 5111 50% 459.5 439.4 544.7 567.7 5249 50% 14 176.7 21.3 282.9 5581 50% 1.8 12.8 2.7 19.4 50% 7.4 8.4 3.8 17.2 5632 50% 119.4 27.5 145.5 56 50% 112.8 32 139.2 57.5 5634 50% 2.4 23.4 6.7 42.3 50% 7.7 22.8 5.4 38.9 5349 50% 8.9 68.4 8.3 122.8 5411 50% 13.8 60.9 17.4 113.4 total 8 AVC 6/8 positive, AVC 8/8 positive, tauns 2/8 positive tauns 4/8 positive

TABLE 8F Overall summary of both diagnostic tests with less diluted samples AVC Flu A/B NP or Test Tauns Test NS1 Read after 15 minutes True Positives 6 2 6 False negatives 2 6 2 Sensitivity  75%  25%  75% Specificity 100% 100% Read after 30 minutes True Positives 8 4 8 False negatives 0 4 0 Sensitivity 100%  50% 100% Specificity 100% 100%

Tables 8G and 8H summarize the test sensitivity (by comparison of signal strengths) for samples diluted to near the limit of detection of the AVC Flu A/B Test.

TABLE 8G Sensitivity of both diagnostic tests with more diluted samples 15′ 30′ sample# final % tauns AVC tauns AVC 5409   50% 9 9.5 8.4 16.4   50% 5.3 11.5 6.5 23.1 5415  0.13% 12.8 41 17.1 72.3 5111  2.50% 43 33.3 54.6 54.6 5248  1.25% 6.9 15.7 7.9 34.7 5249    5% 4.5 27.1 5.2 48.6 5312  0.13% 6 19.5 7.4 27.5 5581 50.00% 1.8 12.8 2.7 19.4 50.00% 7.4 8.4 3.8 17.2 5632 50.00% 119.4 27.5 145.5 56 50.00% 112.8 32 139.2 57.5 5634 50.00% 2.4 23.4 6.7 42.3 50.00% 7.7 22.8 5.4 38.9 5349 12.50% 6.3 12.5 4 21 5411 12.50% 3.7 7 3.1 16.1 5900    5% 53.2 23.4 72.5 44.2 5325  0.13% 7.6 24.3 8.7 39.4 5336  1.25% 3.6 15.4 5 24.8 5589    4% 13 29.8 19 50.2 5901    5% 10.9 25.9 17.8 42.1 total 16 AVC 12/16 positive, AVC 16/16 positive, tauns 3/16 positive tauuns 6/16 positive

TABLE 8H Overall summary of both diagnostic tests with more diluted samples AVC Flu A/B NP or Test Tauns Test NS1 Read after 15 minutes True Positives 12 3 12 False negatives 4 13 4 Sensitivity  75%  19%  75% Specificity 100% 100% Read after 30 minutes True Positives 16 6 16 False negatives 0 10 0 Sensitivity 100%  38% 100% Specificity 100% 100%

In summary, the AVC Flu AB test using pan-specific antibodies for the NS1 antigen was generally more sensitive in detecting influenza A in clinical samples than NP-directed tests such as the Tauns test and the AVC NP test (Tables 8A-H). However, as also seen from Tables 8A-H above, some samples showed detectable amounts of NP, but not NS1, at 15 and/or 30 minutes. Thus, combining an NS1 assay such as the AVC Flu A/B test with an NP assay increases the overall sensitivity and reliability of diagnosis of infection. Tables 8I and 8J compares the specificity of both diagnostic tests after 15 minutes and 30 minutes respectively. Neither the Tauns NP test nor the AVC Flu A/B test gave any false-positive results with influenza B-infected samples.

TABLE 8I Results after 15 minutes (2-fold dilution) Sample Dilution Sample Tauns NP AVC NS1 ID factor status test test 1655 ½ B-positive − − 3614 ½ B-positive − − 4924 ½ B-positive − − 7145 ½ B-positive − − 7815 ½ B-positive − − 97 ½ negative − − 99 ½ negative − − 100 ½ negative − − 108 ½ negative − − 103 ½ negative − − Flu A Specificity: 100% 100%

TABLE 8J Results after 30 minutes (2-fold dilution) Sample Dilution Sample Tauns NP AVC NS1 ID factor status test test 1655 ½ B-positive − − 3614 ½ B-positive − − 4924 ½ B-positive − − 7145 ½ B-positive − − 7815 ½ B-positive − − 97 ½ negative − − 99 ½ negative − − 100 ½ negative − − 108 ½ negative − − 103 ½ negative − − Flu A Specificity 100% 100%

Example 7 Detection of Influenza B in Clinical Samples Using Antibody-Based Assays for Both NS1 and NP

The AVC Flu A/B test was modified to detect influenza B by using a capture antibody that was pan-specific for influenza B NS1 and a second detection antibody that was similarly pan-specific for influenza B NS1 (hereafter called the “AVC Flu A/B” test). The sensitivity of the influenza B-specific AVC Flu A/B test was compared with sensitivity of the Tauns assay for the NP antigen of influenza B. The sample test conditions and final buffer compositions were as described in Example 6.

Tables 9A and 9B compare the sensitivity of both diagnostic tests after 15 minutes and 30 minutes respectively.

TABLE 9A Results for influenza B samples after 15 minutes Sample Dilution Sample Tauns NP AVC NS1 ID factor status test test 1655 ½ B-positive − + 3614 ½ B-positive − − 4924 ½ B-positive + − 7145 ½ B-positive + + 7815 ½ B-positive + + 97 ½ negative − − 99 ½ negative − − 100 ½ negative − − 108 ½ negative − − 103 ½ negative − − Flu B Sensitivity:  60%  60% Flu B Specificity: 100% 100%

TABLE 9B Results for influenza B samples after 30 minutes Sample Dilution Sample Tauns NP AVC NS1 ID factor status test test 1655 ½ B-positive − + 3614 ½ B-positive − − 4924 ½ B-positive + − 7145 ½ B-positive + + 7815 ½ B-positive + + 97 ½ negative − − 99 ½ negative − − 100 ½ negative − − 108 ½ negative − − 103 ½ negative − − Flu B Sensitivity:  60%  60% Flu B Specificity: 100% 100%

As seen, the AVC Flu A/B Test based on NS1 and BL Tauns Test based on NP have comparable sensitivity for Influenza B. Because the NS1 assay could detect influenza B in some samples that showed negative for the presence of NP, and visa versa, a combined NS1/NP test has increased sensitivity.

Example 8 Disease Prognosis from Absolute and/or Relative Quantities of NS1 and NP

The relative quantities of NS1 and NP were determined at various time points in infected subjects. A throat sample or a nasal sample obtained from individual suspected of having influenza was taken at different days (wherein day 1 represents the day of onset of at least one symptom of influenza, e.g., sore throat, sneezing, runny nose, fever, chills, tiredness, dry cough, headache, body aches and/or stomach symptoms such as nausea, vomiting and diarrhea). The swab was extracted in 3 ml of M4 viral transport media. The sample was tested using the AVC Flu A/B test (for NS1) and an AVC Flu A NP Test (an antibody-based test for influenza A NP). 67 μl of the sample was admixed with 33 μl AVC Flu A/B Buffer. The sample was tested as described in Example 4, and results read after 30 minutes. The test line intensity for both tests was quantified with an instrument, and the log(NS1_intensity/NP_intensity) was calculated. Results are shown in FIGS. 19 and 20. As seen, a higher NS1/NP ratio is observed at earlier stages of infection compared to later stages in both throat (FIG. 19) and nasal (FIG. 20) samples. In both cases, the log ratio went from positive to negative around the third day post infection. FIG. 21 gives CAMAG Absorbance Unit Values for AVC Flu A/B Tests (NS1) and AVC Flu A NP Tests of patients as a function of time.

Example 9 Performance of a Rapid Dual-Antigen (NS1 and NP) Diagnostic Test for Influenza A on Human Clinical Samples

A rapid influenza A diagnostic test based on detection of the NS1 and NP antigens was evaluated using human clinical samples. The test was performed in a lateral flow assay format using different monoclonal antibodies as capture agent and detection agent. In the lateral flow format, the capture agent was deposited (immobilized) on a solid support, e.g., a nitrocellulose membrane, onto an area shaped as a thin stripe, called a “test line.” Two different capture agent formats were evaluated. In one format, the NS1 and NP capture agents were each deposited onto separate test lines (hereafter, the “NS1 and NP” format). In the second format, both NS1 and NP capture agents were deposited onto the same test line (hereafter, the “NS1/NP” format). The assembled dipstick was enclosed in a cassette. The detection agent was a combination of gold conjugated anti-influenza A NS1 monoclonal antibodies as well as a gold-conjugated anti-influenza A NP monoclonal antibody. The different test formats for capture agent are shown schematically in FIG. 18. The second and third format were employed in this Example (FIG. 18, middle and right sections).

In further detail, 100 μl of a clinical sample was added to a tube with Lysis buffer (dried buffer), and after 5-10 minutes, 50 μl of Sample Buffer was added to the tube. The gold-mAb detection agents was added to the treated sample and 100 μl of the final sample was transferred with additives to the cassette well of the lateral flow device. Results were read at 30 minutes.

Samples collected were nasopharyngeal swabs extracted in 3 ml of BD Universal Transport Medium from symptomatic patients in the 2007-2008 Flu season in Northern California were analyzed by PCR (Prodessee RT-PCR assay) for presence or absence of Influenza A. Residual sample was stored frozen and subsequently analyzed by AVC using a prototype dual antigen assay. The dual-antigen assay independently detects NS1 and NP using a monoclonal antibody sandwich assay in a lateral flow strip format. Analysis of sixty Influenza A PCR-positive specimens and sixty PCR-negative specimens was undertaken to understand the relative performance for the two antigens (NS1 and NP).

Test Sensitivity:

Of the 60 PCR positive samples, 37 were positive for both antigens, 7 were positive for NS1 only, and an additional 3 for NP1 only. The two antigens are detected in many of the same Flu A positive samples (i.e. 37) but also each antigen is detected alone in a small but significant number (10) of Flu A positive samples not detected by the other. Thus the use of the two antigens significantly increases the detection of Flu A positive samples over the use of either antigen alone. As summarized in Table 10, the sensitivity of the dual-antigen assay in both the two-line “NS1 or NP” test format and the single-line “NS1/NP” test format is 78%, which is significantly improved over the NS1-alone sensitivity of 73% or the NP-alone sensitivity of 67%.

Test Specificity:

Of the 60 PCR negative samples, 1 was a false positive for both antigens, and three were false positives for the NP antigen alone. This leads to a specificity of 98% for NS1 alone, 93% for NP alone, as well as 93% specificity for “NS1 or NP” and “NS1/NP” test formats.

TABLE 10 AVC Flu A Test Results measuring 60 Influenza A positive samples (PCR method used as reference) 30′ Test Test Lines Counts NS1+NP+ 37 NS1+NP− 7 NS1−NP+ 3 NS1−NS1− 13 (NS1/NP)+ 47 NS1 NP NS1 or NP NS1/NP % Sensitivity, 30^(′) 73 67 78 78 AVC Flu A Test Results measuring 60 Influenza A negative samples (PCR method used as reference) 30′ Test Test Lines Counts NS1+NP+ 1 NS1+NP− 0 NS1−NP+ 3 NS1−NP− 56 (NS1/NP)− 56 NS1 NP NS1 or NP NS1/NP % Specificity, 30′ 98 93 93 93

Example 10 Comparison of a Lateral Flow Assay for Influenza B NS1 with an Immunoassay for NP on Human Clinical Samples

A rapid influenza B diagnostic test based on detection of the influenza B NS1 was devised and its performance evaluated in comparison with a commercially available Influenza A/B rapid test on human B-positive and negative clinical samples. An influenza B NS1 capture monoclonal antibody was striped in a nitrocellulose membrane. The detection system comprised a combination of two gold conjugated anti-influenza B NS1 monoclonal antibodies. Samples were collected and the assay performed as in Example 9. Influenza B positive samples collected were nasopharyngeal swabs extracted in 3 ml of BD Universal Transport Medium from symptomatic patients in the 2007-2008 Flu season in Northern California which tested positive by PCR (Prodessee RT-PCR assay) for presence of Influenza B. Residual sample was stored frozen and analyzed by the influenza B NS1 and the commercially available influenza A/B NP test. Results were read at 15 and 30 minutes.

TABLE 11a Results read at 15 minutes Sample Dilution Sample NP AVC NS1 ID factor status test test 1655 ½ B-positive − + 3614 ½ B-positive − − 4924 ½ B-positive + − 7145 ½ B-positive + + 7815 ½ B-positive + + 97 ½ negative − − 99 ½ negative − − 100 ½ negative − − 108 ½ negative − − 103 ½ negative − − Flu B Sensitivity:  60%  60% Flu B Specificity: 100% 100%

TABLE 11b results read at 30 minutes Sample Dilution Sample NP AVC NS1 ID factor status test test 1655 ½ B-positive − + 3614 ½ B-positive − − 4924 ½ B-positive + − 7145 ½ B-positive + + 7815 ½ B-positive + + 97 ½ negative − − 99 ½ negative − − 100 ½ negative − − 108 ½ negative − − 103 ½ negative − − Flu B Sensitivity:  60%  60% Flu B Specificity: 100% 100%

Test Sensitivity

Of the five influenza B positive samples, 2 were positive for both antigens, one was positive for NS1 alone, one was positive for NP alone, and 2 were negative for NS1 and two were negative for NP. Each antigen is detected alone in one Flu B positive sample not detected by the other. The sensitivity based on NS1 alone or NP alone was 60%. However, the use of the two antigens increases the sensitivity from 60% to 80%. This confirmed that combining the NS1 and NP markers can increase the sensitivity of the diagnostic relative to the individual antigens.

Test Specificity

The overall specificity was 100% for the NS1 and the NP markers (n=5 samples).

Example 11 Performance of a Rapid Dual-Antigen (NS1 and NP) Diagnostic Test for Influenza B on Human Clinical Samples

A dual-antigen test that simultaneously assayed a single sample for NS1 and NP protein of influenza B was designed, similar to that of Example 9. Again, two formats were used. In one format, the NS1 and NP test lines were separated (designated the “NS1 or NP” test), as shown in the middle section of FIG. 18. In a second format, the anti-NS1 and anti-NP antibody captures were deposited together in one line (NS1/NP), as shown in the right section of FIG. 18. The NS1 capture test line contained anti-influenza B NS1 monoclonal antibody striped in a nitrocellulose membrane. The NP capture line contained an anti-influenza B NP monoclonal antibody striped in a nitrocellulose membrane. The membrane was enclosed in a cassette. The detection agent was a combination of gold conjugated anti-influenza B NS1 monoclonal antibodies as well as a gold-conjugated anti-influenza B NP monoclonal antibody.

Samples were collected and the assay performed as in Example 9. Results are shown in FIG. 22.

Relative NS1/NP Test Sensitivity:

Of the 10 PCR positive samples measured, 9 were positive for both antigens. Two of the specimens had similar NS1 and NP line intensities, six of the samples had NS1 line intensities greater than NP line intensities, and one sample had an NP line intensity greater than NS1. These results demonstrate that a combined assay for both NS1 and NP influenza B antigen has improved performance over the NS1 or NP test alone.

Flu B Negative Sample Measurements:

Most of the influenza-negative samples were visually negative at the NS1 and NP lines. Samples N13 and N20 had a false positive line at the visible threshold for NP and NS1 respectively. Sample N20 had a readily visible line at the NP line.

These results indicate that an immuno-diagnostic assay detecting the NS1 and NP antigens from influenza A (or B) were superior to a test detecting only one of the antigens (either NS1 or NP alone). For example, the results presented show that the signal strength for NS1 levels in some cases exceeded NP levels, and vice-versa. Thus, samples with overall low viral load favored the dual antigen test in multiple instances, for example where NS1 levels were detectable and NP levels were not, and vice versa. In addition, in some cases where both NS1 and NP levels were so low that neither generated a detectable signal within the detection limit of an assay for either antigen alone, the combined signal strength of both antigens in a single-line NS1/NP format resulted in a combined detection signal above the detection threshold.

Even in cases where NS1 and NP levels were both detectable, the dual antigen test had the additional advantage of allowing the determination of disease stage based on the NS1/NP ratio where the NS1 and NP test lines were separated.

As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to indicate the plural forms as well as the single form. The term “about,” as used herein when referring to a measurable value such as an amount of a compound, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. In addition, if any aspect which is indicated to be optional (e.g., through terminology such as “optionally” or “can” or “for example,” then variations which explicitly exclude the optional feature are also contemplated.

The disclosures of all United States patent references, other publications, GenBank citations and the like cited herein are hereby incorporated by reference to the extent they are consistent with the disclosures herein. If a Genbank citation is associated with more than variant of a sequence, the variant of the sequence assigned the GenBank ID No. at the filing date of the application or priority application (if disclosed in the priority application) is meant. 

1-42. (canceled)
 43. An array comprising at least one first agent that binds to influenza virus non-structural 1 (NS1) protein and at least one agent that binds to influenza virus nucleoprotein (NP) protein.
 44. The array of claim 43, wherein at least one NS1-binding agent and/or at least one NP-binding agent is an antibody or a Post synaptic density protein 95 (PSD95)/Drosophila discs large tumor suppressor (DlgA)/Zonula occludens 1 protein (ZO1) (PDZ) polypeptide.
 45. The array of claim 43, comprising at least two pan-specific NS1 antibodies and/or at least two pan-specific NP antibodies.
 46. The array of claim 43, comprising at least two subtype-specific NS1 antibodies and/or at least two subtype-specific NP antibodies.
 47. The array of claim 43, wherein the array comprises at least one antibody specific for influenza A NS1 and at least one antibody specific for influenza B NS1.
 48. The array of claim 43, wherein the array comprises at least one antibody specific for influenza A NP and at least one antibody specific for influenza B NP.
 49. The array of claim 43, further comprising at least one agent that binds to an influenza virus matrix (M1) protein and at least one agent that binds to an influenza virus hemagglutinin (HA) protein.
 50. A kit for the assessment of an influenza virus infection in a subject, comprising: (a) a first agent for determining the amount of influenza virus non-structural 1 (NS1) protein, and (b) a second agent for determining the amount of influenza virus nucleoprotein (NP) protein.
 51. The kit of claim 50, when the first agent or the second agent comprises at least one antibody.
 52. The array of claim 44, comprising at least two pan-specific NS1 antibodies and/or at least two pan-specific NP antibodies.
 53. The array of claim 44, further comprising at least one agent that binds to an influenza virus matrix (M1) protein and at least one agent that binds to an influenza virus hemagglutinin (HA) protein.
 54. The array of claim 45, further comprising at least one agent that binds to an influenza virus matrix (M1) protein and at least one agent that binds to an influenza virus hemagglutinin (HA) protein.
 55. The array of claim 46, further comprising at least one agent that binds to an influenza virus matrix (M1) protein and at least one agent that binds to an influenza virus hemagglutinin (HA) protein.
 56. The array of claim 47, further comprising at least one agent that binds to an influenza virus matrix (M1) protein and at least one agent that binds to an influenza virus hemagglutinin (HA) protein.
 57. The array of claim 48, further comprising at least one agent that binds to an influenza virus matrix (M1) protein and at least one agent that binds to an influenza virus hemagglutinin (HA) protein. 